Fluorescent protein sensors for measuring the pH of a biological sample

ABSTRACT

Disclosed are fluorescent protein sensors for measuring the pH of a sample, nucleic acids encoding them, and methods of use. The preferred fluorescent protein sensors are variants of the green fluorescent protein (GFP) from  Aequorea victoria . Also disclosed are compositions and methods for measuring the pH of a specific region of a cell, such as the mitochondrial matrix or the Golgi lumen.

This application is a divisional application of U.S. application Ser.No. 09/172,063, filed Oct. 13, 1998, now U.S. Pat. No. 6,150,176, whichis a continuation-in-part of U.S. application Ser. No. 09/094,359, filedJun. 9, 1998, now U.S. Pat. No. 6,140,132. The disclosures of the priorapplications are considered part of and are incorporated by reference inthe disclosure of this application.

This invention was made with Government support under Grant No. NS27177,awarded by the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF THE INVENTION

The invention relates generally to compositions and methods formeasuring the pH of a sample and more particularly to fluorescentprotein sensors for measuring the pH of a biological sample.

BACKGROUND OF THE INVENTION

The pH within various cellular compartments is regulated to provide forthe optimal activity of many cellular processes. In the secretorypathway, posttranslational processing of secretory proteins, thecleavage of prohormones, and the retrieval of escaped luminalendoplasmic reticulum proteins are all pH-dependent.

Several techniques have been described for measuring intracellular pH.Commonly used synthetic pH indicators can be localized to the cytosoland nucleus, but not selectively in organelles other than those in theendocytotic pathway. In addition, some cells are resistant to loadingwith cell-permeant dyes because of physical barriers such as the cellwall in bacteria, yeast, and plants, or the thickness of a tissuepreparation such as brain slices.

Several methods have been described for measuring pH in specific regionsof the cell. One technique uses microinjection of fluorescent indicatorsenclosed in liposomes. Once inside the cell, the liposomes fuse withvesicles in the trans-Golgi, and the pH of the intracellularcompartments is determined by observing the fluorescence of theindicator. This procedure can be laborious, and the fluorescence of theindicator can be diminished due to leakage of the fluorescent indicatorfrom the Golgi, or flux of the fluorescent indicator out of the Golgi aspart of the secretory traffic in the Golgi pathway. In addition, thefusion of the liposomes and components of the Golgi must take place at37□C; however, this temperature facilitates leakage and flux of thefluorescent indicator from the Golgi.

A second method for measuring pH utilizes retrograde transport offluorescein-labeled verotoxin 1B, which stains the entire Golgi complexen route to the endoplasmic reticulum. This method can be used, however,only in cells bearing the receptor globotriaosyl ceramide on the plasmamembrane, and it may be limited by the residence time of the verotoxinin transit through the Golgi.

In a third method, intracellular pH has been measured using the chimericprotein CD25-TGN38, which cycles between the trans-Golgi network and theplasma membrane. At the plasma membrane, the CD25-motif bindsextra-cellular anti-CD25 antibodies conjugated with a pH-sensitivefluorophore. Measurement of fluorescence upon return of the boundcomplex to the Golgi can be used to measure the pH of the organelle.

SUMMARY OF THE INVENTION

The invention is based on the discovery that proteins derived from theAequorea victoria green fluorescence protein (GFP) show reversiblechanges in fluorescence over physiological pH ranges.

Accordingly, in one aspect, the invention provides a method fordetermining the pH of a sample by contacting the sample with anindicator including a first fluorescent protein moiety whose emissionintensity changes as the pH varies between 5 and 10, exciting theindicator, and the determining the intensity at a first wavelength. Theemission intensity of the first fluorescent protein moiety indicates thepH of the sample.

In another aspect, the invention provides a method for determining thepH of a region of a cell by introducing into the cell a polynucleotideencoding a polypeptide including a first fluorescent protein moietywhose emission intensity changes as the pH varies between 5 and 10,culturing the cell under conditions that permit expression of thepolynucleotide, and determining the intensity at a first wavelength. Theemission intensity of the first fluorescent protein moiety indicates thepH of the sample.

In a further aspect, the invention provides a functional engineeredfluorescent protein whose amino acid sequence is substantially identicalto the amino acid sequence of the 238 amino acid Aequorea victoria greenfluorescence protein shown in FIG. 3 (SEQ ID NO:2) of U.S. applicationSer. No. 08/911,825, now issued U.S. Pat. No. 6,054,321, and whoseemission intensity changes as pH varies between 5 and 10.

In another aspect, the invention provides a polynucleotide encoding thefunctional engineered fluorescent protein.

The invention also includes a kit useful for the detection of pH in asample, e.g., a region of a cell. The kit includes a carrier meanscontaining one or more containers comprising a first containercontaining a polynucleotide encoding a polypeptide including a firstfluorescent protein moiety whose emission intensity changes as the pHvaries between 5 and 10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram depicting fluorescent protein sensors usedas indicators of intracellular pH.

FIGS. 2A and 2B are graphs showing absorbance as a function ofwavelength for the fluorescent protein pH sensor EYFP (SEQ ID NO:4; seeExamples 1 and 11 for abbreviations) at various wavelengths (FIG. 2A),and the pH dependency of fluorescence of various GFP fluorescent proteinsensors in vitro and in cells (FIG. 2B). The fluorescence intensity ofpurified recombinant GFP mutant protein (solid symbols) as a function ofpH was measured in a microplate fluorometer. The fluorescence of theGolgi region of HeLa cells expressing proteins having the 81 N-terminalamino acids of the type II membrane-anchored proteingalactosyltransferase (GT:UDP-galactose-β,1,4-galactosyltransferase. EC2.4.1.22) (“GT”) fused to EYFP, or EGFP, i.e., GT-EYFP or GT-EGFP (opensymbols) was determined during pH titration with the ionophoresmonensin/nigericin in high KCL solutions.

FIGS. 3A and 3B are graphs showing ratiometric measurements of pH_(G) bycotransfecting HeLa cells with polynucleotides encoding GT-ECFP andGT-EYFP. FIG. 3A is a graph showing single wavelength fluorescenceintensities of GT-EYFP and GT-ECFP in the Golgi region of a HeLa cell.FIG. 3B is a graph showing the ratio of GT-EYFP/GT-ECFP fluorescence inthe same cell as a function of time.

FIG. 4 is a graph showing the change in mitochondrial pH of HeLa cellsexpressing YFP H148G.

FIG. 5 is a graph showing the mitochondrial pH of chick skeletalmyotubes expressing YFP H148G in the mitochondrial matrix.

FIG. 6 is a graph showing fluorescence and pH in HeLa cells expressingYFP H148Q targeted to the mitochondrial matrix.

FIG. 7 is a graph showing a ratiometric measurement of mitochondrial pHfollowing expression of YFP H148G (pH sensitive) and GFP T203I (pHinsensitive) in mitochondria of HeLa cells.

FIG. 8 is a graph showing normalized absorbance of WT GFP and YFP in 75mM phosphate pH 8.0, 140 mM NACl. Solid lines, WT GFP; Dashed lines,YFP.

FIG. 9 is a stereoview of the 2FO-FC electron density map of the YFPchromophore and the stacked Tyr203 after refinement. The 2.5 Åresolution map was contoured at +1 standard deviation.

DETAILED DESCRIPTION

The invention provides genes encoding fluorescent sensor proteins, orfragments thereof, whose fluorescence is sensitive to changes in pH at arange between 5 and 10. The proteins of the invention are useful formeasuring the pH of a sample. The sample can be a bioloigical sample andcan include an intracellular region of a cell, such as the lumen of themitochondria or golgi. The pH of a sample is determined by observing thefluorescence of the fluorescent sensor protein.

The fluorescent protein pH sensor have a broad applicability to cellsand organisms that are amenable to gene transfer. Problems associatedwith the use of other agents used to measure pH, e.g., problemsassociated with permeabilizing cells to ester-containing agents, leakageof agents, or hydrolysis of agents are avoided. With the fluorescentprotein pH sensors of the invention, no leakage occurs over the courseof a typical measurement, even when the measurement is made at 37° C.

Compositions and methods described herein also avoid the need to expressand purify large quantities of soluble recombinant protein, purify andlabel it in vitro, microinject it back into cells. An importantadvantage of the fluorescent protein pH sensors of the invention is thatthey can be delivered to cells in the form of polynucleotides encodingthe protein sensor fused to a targeting signal or signals. The targetingsignal directs the expression of the protein sensors to restricted celllocations. Thus, it is possible to measure the pH of a precisely definedcellular region or organelle.

Polynucleotides and Polypeptides

In a first aspect, the invention provides a functional engineeredfluorescent protein whose amino acid sequence is substantially identicalto the 238 amino acid Aequorea victoria green fluorescence protein shownin FIG. 3 (SEQ ID NO:2) of U.S. application Ser. No. 08/911,825, nowissued U.S. Pat. No. 6,054,321. The term “fluorescent protein” refers toany protein capable of emitting light when excited with appropriateelectromagnetic radiation, and which has an amino acid sequence that iseither natural or engineered and is derived from the amino acid sequenceof Aequorea-related fluorescent protein. The term “fluorescent proteinpH sensor” refers to a fluorescent protein whose emitted light varieswith changes in pH from 5 to 10.

The invention also includes functional polypeptide fragments of afluorescent protein pH sensor. As used herein, the term “functionalpolypeptide fragment” refers to a polypeptide which possesses biologicalfunction or activity which is identified through a defined functionalassay and which is associated with a particular biologic, morphologic,or phenotypic alteration in the cell. The term “functional fragments ofa functional engineered fluorescent protein” refers to fragments of afunctional engineered protein that retain a function of the engineeredfluorescent protein, e.g., the ability to fluoresce in a pH-dependentmanner over the pH range 5 to 10. Biologically functional fragments canvary in size from a polypeptide fragment as small as an epitope to alarge polypeptide.

Minor modifications of the functional engineered fluorescent protein mayresult in proteins which have substantially equivalent activity ascompared to the unmodified counterpart polypeptide as described herein.Such modifications may be deliberate, as by site-directed mutagenesis,or may be spontaneous. All of the polypeptides produced by thesemodifications are included herein as long as the pH-dependentfluorescence of the engineered protein still exists.

A functional engineered fluorescent protein includes amino acidsequences substantially the same as the sequence set forth in SEQ IDNO:2, and whose emission intensity changes as pH varies between 5 and10. In some embodiments the emission intensity of the functionalengineered fluorescent protein changes as pH varies between 5 and 8.5.

By “substantially identical” is meant a protein or polypeptide thatretains the activity of a functional engineered protein, or nucleic acidencoding the same, and which exhibits at least 80%, preferably 85%, morepreferably 90%, and most preferably 95% homology to a reference aminoacid or nucleic acid sequence. For polypeptides, the length ofcomparison sequences will generally be at least 16 amino acids,preferably at least 20 amino acids, more preferably at least 25 aminoacids, and most preferably 35 amino acids. For nucleic acids, the lengthof comparison sequences will generally be at least 50 nucleotides,preferably at least 60 nucleotides, more preferably at least 75nucleotides, and most preferably 110 nucleotides.

By “substantially identical” is meant an amino acid sequence whichdiffers only by conservative amino acid substitutions, for example,substitution of one amino acid for another of the same class (erg.,valine for glycine, arginine for lysine, etc.) or by one or morenon-conservative substitutions, deletions, or insertions located atpositions of the amino acid sequence which do not destroy the functionof the protein (assayed, e.g., as described herein). Preferably, such asequence is at least 85%, more preferably 90%, more preferably 95%, morepreferably 98%, and most preferably 99% identical at the amino acidlevel to one of the sequences of EGFP (SEQ ID NO:3), EYFP (SEQ ID NO:4),ECFP (SEQ ID NO: 6), EYFP-V68L/Q69K (SEQ ID NO:5), YFP H148G (SEQ IDNO:7), or YFP H148Q (SEQ ID NO:8).

Homology is typically measured using sequence analysis software (e.g.,Sequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Avenue,Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various substitutions, deletions,insertions, and other modifications. Conservative substitutionstypically include substitutions within the following groups: glycinealanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine.

In some embodiments, the amino acid sequence of the protein includes oneof the following sets of substitutions in the amino acid sequence of theAequorea green fluorescent protein (SEQ ID NO:2): F64L/S65T/H231L,referred to herein as EGFP (SEQ ID NO:3); S65G/S72A/T203Y/H231L,referred to herein as EYFP (SEQ ID NO:4);S65G/V68L/Q69K/S72A/T203Y/H231L, referred to herein as EYFP-V68L/Q69K(SEQ ID NO:5); K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L,referred to herein as ECFP (SEQ ID NO:6). The amino acid sequences ofEGFP, EYFP, ECFP, and EYFP-V68L/Q69K are shown in Tables 1-4,respectively. The amino acids are numbered with the amino acid followingthe initiating methionine assigned the ‘1’ position. Thus, F64Lcorresponds to a substitution of leucine for phenylalanine in the 64thamino acid following the initiating methionine.

TABLE 1 EGFP Amino Acid Sequence (SEQ ID NO:3)MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

TABLE 2 EYFP Amino Acid Sequence (SEQ ID NO:4)MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

TABLE 3 EYFP-V68L/Q69K Amino Acid Sequence (SEQ ID NO:5)MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

TABLE 4 ECFP Amino Acid Sequence (SEQ ID NO:6)MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCESRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKAHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

In other embodiments, the amino acid sequence of the protein is based onthe sequence of the wild-type Aequorea green fluorescent protein, butincludes the substitution H148G (SEQ ID NOs:7 and 9) or H148Q (SEQ IDNOs:8 and 10). In specific embodiments, these substitutions can bepresent along with other substitutions, e.g., the proteins can includethe substitutions S65G/V68L/S72A/Q80R/H148G/T203Y, which is referred toherein as the YFP H148G mutant ((SEQ ID NO:7);S65G/V68L/S72A/Q80R/H148Q/T203Y, which is referred to herein as the YFPH148Q mutant (SEQ ID NO:8); as well as S65G/S72A/H148G/T203Y/H231L,which is referred to herein as EYFP-H148G (SEQ If) NO:9) andS65G/S72A/H148Q/T203Y/H231L, which is referred to herein as EYFP-H148Q(SEQ ID NO:10). The amino acid sequences of these mutants are shown inTables 5-8, respectively.

TABLE 5 Amino Acid Sequence of YFP H148G (SEQ ID NO:7)MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSGNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

TABLE 6 Amino Acid Sequence of YFP H148Q (SEQ ID NO:8)MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSQNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

TABLE 7 Amino Acid Sequence of EYFP-H148G (SEQ ID NO:9)MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSGNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

TABLE 8 Amino acid Sequence of EYFP-H148Q (SEQ ID NO:10)MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSQNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

In some embodiments, the protein or polypeptide is substantiallypurified. By “substantially pure protein or polypeptide” is meant anfunctional engineered fluorescent polypeptide which has been separatedfrom components which naturally accompany it. Typically, the protein orpolypeptide is substantially pure when it is at least 60%, by weight,free from the proteins and naturally-occurring organic molecules withwhich it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, of the protein. A substantially pure protein may beobtained, for example, by extraction from a natural source (e.g., aplant cell); by expression of a recombinant nucleic acid encoding afunctional engineered fluorescent protein; or by chemically synthesizingthe protein. Purity can be measured by any appropriate method, e.g.,those described in column chromatography, polyacrylamide gelelectrophoresis, or by HPLC analysis.

A protein or polypeptide is substantially free of naturally associatedcomponents when it is separated from those contaminants which accompanyit in its natural state. Thus, a protein or polypeptide which ischemically synthesized or produced in a cellular system different fromthe cell from which it naturally originates will be substantially freefrom its naturally associated components. Accordingly, substantiallypure polypeptides include those derived from eukaryotic organisms butsynthesized in E. coli or other prokaryotes.

The invention also provides polynucleotides encoding the functionalengineered fluorescent protein described herein. These polynucleotidesinclude DNA, cDNA, and RNA sequences which encode functional engineeredfluorescent proteins. It is understood that all polynucleotides encodingfunctional engineered fluorescent proteins are also included herein, aslong as they encode a protein or polypeptide whose fluorescent emissionintensity changes as pH varies between 5 and 10. Such polynucleotidesinclude naturally occurring, synthetic, and intentionally manipulatedpolynucleotides. For example, the polynucleotide may be subjected tosite-directed mutagenesis. The polynucleotides of the invention includesequences that are degenerate as a result of the genetic code.Therefore, all degenerate nucleotide sequences are included in theinvention as long as the amino acid sequence of the functionalengineered fluorescent protein or derivative is functionally unchanged.

Specifically disclosed herein is a polynucleotide sequence encoding afunctional engineered fluorescent protein that includes one of thefollowing sets of substitutions in the amino acid sequence of theAequorea green fluorescent protein (SEQ ID NO:2): S65G/S72A/T203Y/H231L,S65G/V68L/Q69K/S72A/T203Y/H231L, orK26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L. In specificembodiments, the DNA sequences encoding EGFP, EYFP, ECFP,EYFP-V68L/Q69K, YFP H148G, YFP H148Q, EYFP-H148G and EYFP-148Q are thoseshown in Tables 9-16 (SEQ ID NOs:11 to 18), respectively.

The nucleic acid encoding functional engineered fluorescent proteins maybe reflect the codon choice in the native A. victoria coding sequence,or, alternatively, may be chosen to reflect the optimal codonfrequencies used in the organism in which the proteins will beexpressed. Thus, nucleic acids encoding a target functional engineeredprotein to be expressed in a human cell may have use a codon choice thatis optimized for mammals, or especially humans.

TABLE 9 EGFP Nucleic Acid Sequence (SEQ ID NO:11)ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCCAGGTCAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 10 EYFP Nucleic Acid Sequence (SEQ ID NO:12)ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 11 ECFP Nucleic Acid Sequence (SEQ ID NO:13)ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAGGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGTACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 12 EYFP-V68L/Q69K Nucleic Acid Sequence (SEQ ID NO:14)ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGAAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 13 Nucleotide Sequence of the YFP-H148G Coding Region (SEQ IDNO:15) ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGGACTACTCCAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTCTTCAATGCTTTGCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTTCAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCAGGCAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCTATCAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA

TABLE 14 Nucleotide Sequence of the YFP H148Q Coding Region (SEQ IDNO:16) ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTCTTCAATGCTTTGCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTTCAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCAGGCAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCTATCAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA

TABLE 15 Nucleotide Sequence of the EYFP-H148G Coding Region (SEQ IDNO:17) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCGGCAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 16 Nucleotide Sequence of the EYFP-H148Q Coding Region (SEQ IDNO:18) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCAGAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

The term “polynucleotide” refers to a polymeric form of nucleotides ofat least 10 bases in length. The nucleotides can be ribonucleotides,deoxyribonucleotides, or modified forms of either type of nucleotide.The term includes single and double stranded forms of DNA. By “isolatedpolynucleotide” is meant a polynucleotide that is not immediatelycontiguous with both of the coding sequences with which it isimmediately contiguous (one on the 5′ end and one on the 3′ end) in thenaturally occurring genome of the organism from which it is derived. Theterm therefore includes, for example, a recombinant DNA which isincorporated into a vector, e.g., an expression vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences.

A “substantially identical” nucleic acid sequence codes for asubstantially identical amino acid sequence as defined above.

The functional engineered fluorescent protein can also include atargeting sequence to direct the fluorescent protein to particularcellular sites by fusion to appropriate organellar targeting signals orlocalized host proteins. A polynucleotide encoding a targeting sequencecan be ligated to the 5′ terminus of a polynucleotide encoding thefluorescence such that the targeting peptide is located at the aminoterminal end of the resulting fusion polynucleotide/polypeptide. Thetargeting sequence can be, e.g., a signal peptide. In the case ofeukaryotes, the signal peptide is believed to function to transport thefusion polypeptide across the endoplasmic reticulum. The secretoryprotein is then transported through the Golgi apparatus, into secretoryvesicles and into the extracellular space or, preferably, the externalenvironment. Signal peptides which can be utilized according to theinvention include pre-pro peptides which contain a proteolytic enzymerecognition site. Other signal peptides with similar properties topro-calcitonin described herein are known to those skilled in the art,or can be readily ascertained without undue experimentation.

The targeting sequence can also be a nuclear localization sequence, anendoplasmic reticulum localization sequence, a peroxisome localizationsequence, a mitochondrial localization sequence, or a localized protein.Targeting sequences can be targeting sequences which are described, forexample, in “Protein Targeting”, chapter 35 of Stryer, L., Biochemistry(4th ed.). W. H. Freeman, 1995. The localization sequence can also be alocalized protein. Some important targeting sequences include thosetargeting the nucleus (KKKRK) (SEQ ID NO:35), mitochondrion (the 12amino terminal amino acids of the cytochrome c oxidase subunit IV gene,or the amino terminal sequence MLRTSSLFTRRVQPSLFRNILRLQST (SEQ IDNO:36), endoplasmic reticulum (KDEL (SEQ ID NO:37) at the C-terminus,assuming a signal sequence present at N-terminus), peroxisome (SKF atC-terminus), prenylation or insertion into plasma membrane (CaaX, CC,CXC, or CCXX at C-terminus), cytoplasmic side of plasma membrane (fusionto SNAP-25), or the Golgi apparatus (fusion to the amino terminal 81amino acids of human type II membrane-anchored proteingalactosyltransferase, or fusion to furin).

Examples of targeting sequences linked to functional engineeredfluorescent proteins include GT-EYFP (SEQ ID NO:22), GT-ECFP, GT-EGFP(SEQ ID NO:21), and GT-EYFP-V68L/Q69K, which are targeted to the Golgiapparatus using sequences from the GT protein; and mito-ECFP (SEQ IDNO:19) and mito-EYFP (SEQ ID NO:20), which are targeted to themitochondrial matrix using sequences from the amino terminal region ofthe cytochrome c oxidase subunit IV gene. The EYFP, ECFP, EGFP, andEYFP-V68L/Q69K amino acid sequences, as well as nucleic acids encodingthese polypeptides, are described above. The GT-derived targetingsequence corresponds to the 81 amino terminal amino acids of the humanGT sequence. The GT amino acid sequences, and the polynucleotidesequences encoding the GT amino acid sequences, are described in GenbankAccession No. M70427 and Mengle-Gaw et al., Biochem. Biophys. Res.Commun. 176 (3), 1269-1276 (1991).

Amino acid sequences of mito-ECFP, mito-EYFP, GT-EGFP, GT-EYFP, mito-YFPH148G, mito-YFP H148Q, mito-EYFP H148G, mito-EYFP-H148Q are shown inTables 17-24.

In specific embodiments, nucleic acid sequences encoding targetingsequences linked to functional engineered fluorescent proteins have thesequences shown in Tables 25-32.

TABLE 17 mito-ECFP Amino Acid Sequence (SEQ ID NO:19)MLSLRQSIRFFKRSGIMVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKAHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

TABLE 18 mito-EYFP Amino Acid Sequence (SEQ ID NO:20)MLSLRQSIREEKRSGIMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSANPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

TABLE 19 GT-EGFP Amino Acid Sequence (SEQ ID NO:21)MRLREPLLSGAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGAMDPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDE LYK

TABLE 20 GT-EYFP Amino Acid Sequence (SEQ ID NO:22)MRLREPLLSGAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGAMDPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDE LYK*

TABLE 21 mito-YFP-H148G Amino Acid Sequence (SEQ ID NO:23)MLRTSSLFTRRVQPSLFRNILRLQSTSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSGNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITH GMDELYK

TABLE 22 mito-YFP-H148Q Amino Acid Sequence (SEQ ID NO:24)MLRTSSLFTRRVQPSLFRNILRLQSTSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSQNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITH GMDELYK

TABLE 23 mito-EYFP-H148G Amino Acid Sequence (SEQ ID NO:25)MLRTSSLFTRRVQPSLFRNILRLQSTMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSGNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

TABLE 24 mito-EYFP-H148Q Amino Acid Sequence (SEQ ID NO:26)MLRTSSLFTRRVQPSLFRNILRLQSTMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSQNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

TABLE 25 GT-ECFP Nucleic Acid Sequence (SEQ ID NO:27)ATGAGGCTTCGGGAGCCGCTCCTGAGCGGCGCCGCGATGCCAGGCGCGTCCCTACAGCGGGCCTGCCGCCTGCTCGTGGCCGTCTGCGCTCTGCACCTTGGCGTCACCCTCGTTTACTACCTGGCTGGCCGCGACCTGAGCCGCCTGCCCCAACTGGTCGGAGTCTCCACACCGCTGCAGGGCGGCTCGAACAGTGCCGCCGCCATCGGGCAGTCCTCCGGGGAGCTCCGGACCGGAGGGGCCATGGATCCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAGGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGTACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 26 mito-EYFP Nucleic Acid Sequence (SEQ ID NO:28)ATGCTGAGCCTGCGCCAGAGCATCCGCTTCTTCAAGCGCAGCGGCATCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 27 GT-EGFP Nucleic Acid Sequence (SEQ ID NO:29)ATGAGGCTTCGGGAGCCGCTCCTGAGCGGCGCCGCGATGCCAGGCGCGTCCCTACAGCGGGCCTGCCGCCTGCTCGTGGCCGTCTGCGCTCTGCACCTTGGCGTCACCCTCGTTTACTACCTGGCTGGCCGCGACCTGAGCCGCCTGCCCCAACTGGTCGGAGTCTCCACACCGCTGCAGGGCGGCTCGAACAGTGCCGCCGCCATCGGGCAGTCCTCCGGGGAGCTCCGGACCGGAGGGGCCATGGATCCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 28 GT-EYFP Nucleic Acid Sequence (SEQ ID NO:30)ATGAGGCTTCGGGAGCCGCTCCTGAGCGGCGCCGCGATGCCAGGCGCGTCCCTACAGCGGGCCTGCCGCCTGCTCGTGGCCGTCTGCGCTCTGCACCTTGGCGTCACCCTCGTTTACTACCTGGCTGGCCGCGACCTGAGCCGCCTGCCCCAACTGGTCGGAGTCTCCACACCGCTGCAGGGCGGCTCGAACAGTGCCGCCGCCATCGGGCAGTCCTCCGGGGAGCTCCGGACCGGAGGGGCCATGGATCCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 29 mito-YFP H148G Nucleic Acid Sequence (SEQ ID NO:31)ATGCTGAGCCTGCGCCAGAGCATCCGCTTCTTCAAGCGCAGCGGCATCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTCTTCAATGCTTTGCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTTCAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCAGGCAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCTATCAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA

TABLE 30 mito-YFP H148Q Nucleic Acid Sequence (SEQ ID NO:32)ATGCTGAGCCTGCGCCAGAGCATCCGCTTCTTCAAGCGCAGCGGCATCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTCTTCAATGCTTTGCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTTCAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCAGGCAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCTATCAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA

TABLE 31 mito-EYFP-H148G Nucleic Acid Sequence (SEQ ID NO:33)ATGCTGAGCCTGCGCCAGAGCATCCGCTTCTTCAAGCGCAGCGGCATCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCGGCAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TABLE 32 mito-EYFP-H148Q Nucleic Acid Sequence (SEQ ID NO:34)ATGCTGAGCCTGCGCCAGAGCATCCGCTTCTTCAAGCGCAGCGGCATCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCAGAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

The fluorescent indicators can be produced as proteins fused to otherfluorescent indicators or targeting sequences by recombinant DNAtechnology. Recombinant production of fluorescent proteins involvesexpressing nucleic acids having sequences that encode the proteins.Nucleic acids encoding fluorescent proteins can be obtained by methodsknown in the art. For example, a nucleic acid encoding the protein canbe isolated by polymerase chain reaction of cDNA from A. victoria usingprimers based on the DNA sequence of A. victoria green fluorescentprotein. PCR methods are described in, for example, U.S. Pat. No.4,683,195; Mullis, et al. Cold Spring Harbor Symp. Quant. Biol. 51:263(1987), and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).Mutant versions of fluorescent proteins can be made by site-specificmutagenesis of other nucleic acids encoding fluorescent proteins, or byrandom mutagenesis caused by increasing the error rate of PCR of theoriginal polynucleotide with 0.1 mM MnCl₂ and unbalanced nucleotideconcentrations. See, e.g., U.S. patent application Ser. No. 08/337,915,filed Nov. 10, 1994, now issued U.S. Pat. No. 5,625,048, orInternational Application PCT/US95/14692, filed Nov. 10, 1995, nowpublished PCT WO 96/23810.

The construction of expression vectors and the expression of genes intransfected cells involves the use of molecular cloning techniques alsowell known in the art. Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,(Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., most recent Supplement).

Nucleic acids used to transfect cells with sequences coding forexpression of the polypeptide of interest generally will be in the formof an expression vector including expression control sequencesoperatively linked to a nucleotide sequence coding for expression of thepolypeptide. As used herein, “operatively linked” refers to ajuxtaposition wherein the components so described are in a relationshippermitting them to function in their intended manner. A control sequenceoperatively linked to a coding sequence is ligated such that expressionof the coding sequence is achieved under conditions compatible with thecontrol sequences. “Control sequence” refers to polynucleotide sequenceswhich are necessary to effect the expression of coding and non-codingsequences to which they are ligated. Control sequences generally includepromoter, ribosomal binding site, and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, components whose presence can influence expression, and canalso include additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

As used herein, the term “nucleotide sequence coding for expression of”a polypeptide refers to a sequence that, upon transcription andtranslation of mRNA, produces the polypeptide. This can includesequences containing, e.g., introns. As used herein, the term“expression control sequences” refers to nucleic acid sequences thatregulate the expression of a nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus, expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalsfor introns, maintenance of the correct reading frame of that gene topermit proper translation of the mRNA, and stop codons.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the fluorescent indicator codingsequence and appropriate transcriptional/translational control signals.These methods include in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. (See, forexample, the techniques described in Maniatis, et al., Molecular CloningA Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989).Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method byprocedures well known in the art. Alternatively, MgCl₂ or RbCl can beused. Transformation can also be performed after forming a protoplast ofthe host cell or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransfected with DNA sequences encoding the fusion polypeptide of theinvention, and a second foreign DNA molecule-encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein. (Eukaryotic Viral Vectors,Cold Spring Harbor Laboratory, Gluzman ed., 1982).

Techniques for the isolation and purification of polypeptides of theinvention expressed in prokaryotes or eukaryotes may be by anyconventional means such as, for example, preparative chromatographicseparations and immunological separations such as those involving theuse of monoclonal or polyclonal antibodies or antigen.

A variety of host-expression vector systems may be utilized to expressfluorescent indicator coding sequence. These include but are not limitedto microorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining a fluorescent indicator coding sequence; yeast transformedwith recombinant yeast expression vectors containing the fluorescentindicator coding sequence; plant cell systems infected with recombinantvirus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobaccomosaic virus, TMV) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing a fluorescent indicator codingsequence; insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus) containing a fluorescent indicator codingsequence; or animal cell systems infected with recombinant virusexpression vectors (e.g., retroviruses, adenovirus, vaccinia virus)containing a fluorescent indicator coding sequence, or transformedanimal cell systems engineered for stable expression.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, transcription enhancer elements, transcriptionterminators, etc. may be used in the expression vector (see, e.g.,Bitter, et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and thelike may be used. When cloning in mammalian cell systems, promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the retrovirus long terminalrepeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter)may be used. Promoters produced by recombinant DNA or synthetictechniques may also be used to provide for transcription of the insertedfluorescent indicator coding sequence.

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for thefluorescent indicator expressed. For example, when large quantities ofthe fluorescent indicator are to be produced, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Those which are engineered to contain acleavage site to aid in recovering fluorescent indicator are preferred.In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, Ed. Ausubel, et al., Greene Publish. Assoc. & WileyInterscience, Ch. 13, 1988; Grant, et al., Expression and SecretionVectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987,Acad. Press, N.Y., Vol. 153, pp.516-544, 1987; Glover, DNA Cloning, Vol.1, IRL Press, Wash., D.C., Ch. 3, 1986; and Bitter, Heterologous GeneExpression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad.Press, N.Y., Vol. 152, pp. 673-684, 1987; and The Molecular Biology ofthe Yeast Saccharomyces, Eds. Strathern et al., Cold Spring HarborPress, Vols. I and II, 1982. A constitutive yeast promoter such as ADHor LEU2 or an inducible promoter such as GAL may be used (Cloning inYeast, Ch. 3, R. Rothstein In: DNA Cloning Vol.11, A Practical Approach,Ed. D M Glover, IRL Press, Wash., D.C., 1986). Alternatively, vectorsmay be used which promote integration of foreign DNA sequences into theyeast chromosome.

In cases where plant expression vectors are used, the expression of afluorescent indicator coding sequence may be driven by any of a numberof promoters. For example, viral promoters such as the 35S RNA and 19SRNA promoters of CaMV (Brisson, et al., Nature 310:511-514, 1984), orthe coat protein promoter to TMV (Takamatsu, et al., EMBO J. 6:307-311,1987) may be used; alternatively, plant promoters such as the smallsubunit of RUBISCO (Coruzzi, et al., 1984, EMBO J. 3:1671-1680; Broglie,et al., Science 224:838-843, 1984); or heat shock promoters, e.g.,soybean hsp 17.5-E or hsp 17.3-B (Gurley, et al., Mol. Cell. Biol.6:559-565, 1986) may be used. These constructs can be introduced intoplant cells using Ti plasmids, Ri plasmids, plant virus vectors, directDNA transformation, microinjection, electroporation, etc. For reviews ofsuch techniques see, for example, Weissbach & Weissbach, Methods forPlant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463,1988; and Grierson & Corey, Plant Molecular Biology, 2d Ed., Blackie,London, Ch. 7-9, 1988.

An alternative expression system which could be used to expressfluorescent indicator is an insect system. In one such system,Autographa californica nuclear polyhedrosis virus (AcNPV) is used as avector to express foreign genes. The virus grows in Spodopterafrugiperda cells. The fluorescent indicator coding sequence may becloned into non-essential regions (for example, the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (for example thepolyhedrin promoter). Successful insertion of the fluorescent indicatorcoding sequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed, see Smith, et al., J. Viol. 46:584, 1983;Smith, U.S. Pat. No. 4,215,051.

Eukaryotic systems, and preferably mammalian expression systems, allowfor proper post-translational modifications of expressed mammalianproteins to occur. Eukaryotic cells which possess the cellular machineryfor proper processing of the primary transcript, glycosylation,phosphorylation, and, advantageously secretion of the gene productshould be used as host cells for the expression of fluorescentindicator. Such host cell lines may include but are not limited to CHO,VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and W138. Primary celllines, such as neonatal rat myocytes, can also be used.

Mammalian cell systems which utilize recombinant viruses or viralelements to direct expression may be engineered. For example, when usingadenovirus expression vectors, the fluorescent indicator coding sequencemay be ligated to an adenovirus transcription/translation controlcomplex, e.g., the late promoter and tripartite leader sequence. Thischimeric gene may then be inserted in the adenovirus genome by in vitroor in vivo recombination. Insertion in a non-essential region of theviral genome (e.g., region E1 or E3) will result in a recombinant virusthat is viable and capable of expressing the fluorescent indicator ininfected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:3655-3659, 1984).: Alternatively, the vaccinia virus 7.5K promoter maybe used (e.g., see, Mackett, et al., Proc. Natl. Acad. Sci. USA ,79:7415-7419, 1982; Mackett, et al., J. Virol. 49: 857-864, 1984; Panicali,et al., Proc. Natl. Acad. Sci. USA 79: 4927-4931, 1982). Of particularinterest are vectors based on bovine papilloma virus which have theability to replicate as extrachromosomal elements (Sarver, et al., Mol.Cell. Biol. 1: 486, 1981). Shortly after entry of this DNA into mousecells, the plasmid replicates to about 100 to 200 copies per cell.Transcription of the inserted cDNA does not require integration of theplasmid into the host's chromosome, thereby yielding a high level ofexpression. These vectors can be used for stable expression by includinga selectable marker in the plasmid, such as the neo gene. Alternatively,the retroviral genome can be modified for use as a vector capable ofintroducing and directing the expression of the fluorescent indicatorgene in host cells (Cone & Mulligan, Proc. Natl. Acad. Sci. USA,81:6349-6353, 1984). High level expression may also be achieved usinginducible promoters, including, but not limited to, the metallothioneinIIA promoter and heat shock promoters.

The recombinant nucleic acid can be incorporated into an expressionvector including expression control sequences operatively linked to therecombinant nucleic acid. The expression vector can be adapted forfunction in prokaryotes or eukaryotes by inclusion of appropriatepromoters, replication sequences, markers, etc.

DNA sequences encoding the fluorescence indicator polypeptide of theinvention can be expressed in vitro or in vivo by DNA transfer into asuitable recombinant host cell. As used herein, “recombinant host cells”are cells in which a vector can be propagated and its DNA expressed. Theterm also includes any progeny of the subject host cell. It isunderstood that all progeny may not be identical to the parental cellsince there may be mutations that occur during replication. However,such progeny are included when the term “recombinant host cell” is used.Methods of stable transfer, in other words when the foreign DNA iscontinuously maintained in the host, are known in the art.

The expression vector can be transfected into a host cell for expressionof the recombinant nucleic acid. Recombinant host cells can be selectedfor high levels of expression in order to purify the fluorescentindicator fusion protein. E. coli is useful for this purpose.Alternatively, the host cell can be a prokaryotic or eukaryotic cellselected to study the activity of an enzyme produced by the cell. Inthis case, the linker peptide is selected to include an amino acidsequence recognized by the protease. The cell can be, e.g., a culturedcell or a cell taken in vivo from a transgenic animal.

Transgenic Animals

In another embodiment, the invention provides a transgenic non-humananimal,that expresses a polynucleotide sequence which encodes afluorescent protein pH sensor.

The “non-human animals” of the invention comprise any non-human animalhaving a polynucleotide sequence which encodes a fluorescent indicator.Such non-human animals include vertebrates such as rodents, non-humanprimates, sheep, dog, cow, pig, amphibians, and reptiles. Preferrednon-human animals are selected from the rodent family including rat andmouse, most preferably mouse. The “transgenic non-human animals” of theinvention are produced by introducing “transgenes” into the germline ofthe non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thezygote is the best target for micro-injection. In the mouse, the malepronucleus reaches the size of approximately 20 micrometers in diameterwhich allows reproducible injection of 1-2 pl of DNA solution. The useof zygotes as a target for gene transfer has a major advantage in thatin most cases the injected DNA will be incorporated into the host genebefore the first cleavage (Brinster et al., Proc. Natl. Acad. Sci. USA82:4438-4442, 1985). As a consequence, all cells of the transgenicnon-human animal will carry the incorporated transgene. This will ingeneral also be reflected in the efficient transmission of the transgeneto offspring of the founder since 50% of the germ cells will harbor thetransgene. Microinjection of zygotes is the preferred method forincorporating transgenes in practicing the invention.

The term “transgenic” is used to describe an animal which includesexogenous genetic material within all of its cells. A “transgenic”animal can be produced by cross-breeding two chimeric animals whichinclude exogenous genetic material within cells used in reproduction.Twenty-five percent of the resulting offspring will be transgenic, i.e.,animals which include the exogenous genetic material within all of theircells in both alleles. 50% of the resulting animals will include theexogenous genetic material within one allele and 25% will include noexogenous genetic material.

Retroviral infection can also be used to introduce transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retro viral infection (Jaenisch, R., Proc. Natl. Acad. SciUSA 73:1260-1264, 1976). Efficient infection of the blastomeres isobtained by enzymatic treatment to remove the zona pellucida (Hogan, etal. (1986) in Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The viral vector systemused to introduce the transgene is typically a replication-defectiveretrovirus carrying the transgene (Jahner, et al., Proc. Natl. Acad.Sci. USA 82:6927-6931, 1985; Van der Putten, et al., Proc. Natl. Acad.Sci USA 82:6148-6152, 1985). Transfection is easily and efficientlyobtained by culturing the blastomeres on a monolayer of virus-producingcells (Van der Putten, supra; Stewart, et al., EMBO J. 6:383-388, 1987).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (D. Jahner etal., Nature 298:623-628, 1982). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic nonhuman animal. Further, the founder maycontain various retro viral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline, albeit with low efficiency, by intrauterine retro viral infectionof the midgestation embryo (D. Jahner et al., supra). A third type oftarget cell for transgene introduction is the embryonal stem cell (ES).ES cells are obtained from pre-implantation embryos cultured in vitroand fused with embryos (M. J. Evans et al. Nature 292:154-156, 1981; M.O. Bradley et al., Nature 309: 255-258,1984; Gossler, et al., Proc.Natl. Acad. Sci USA 83: 9065-9069, 1986; and Robertson et al., Nature322:445-448, 1986). Transgenes can be efficiently introduced into the EScells by DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anonhuman animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. (Forreview see Jaenisch, R., Science 240: 1468-1474, 1988).

“Transformed” means a cell into which (or into an ancestor of which) hasbeen introduced, by means of recombinant nucleic acid techniques, aheterologous polynucleotide. “Heterologous” refers to a polynucleotidesequence that either originates from another species or is modified fromeither its original form or the form primarily expressed in the cell.

“Transgene” means any piece of DNA which is inserted by artifice into acell, and becomes part of the genome of the organism (i.e., eitherstably integrated or as a stable extrachromosomal element) whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Included within this definition is a transgene created bythe providing of an RNA sequence which is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences which encode the fluorescent indicator which may beexpressed in a transgenic non-human animal. The term “transgenic” asused herein additionally includes any organism whose genome has beenaltered by in vitro manipulation of the early embryo or fertilized eggor by any transgenic technology to induce a specific gene knockout. Theterm “gene knockout” as used herein, refers to the targeted disruptionof a gene in vivo with complete loss of function that has been achievedby any transgenic technology familiar to those in the art. In oneembodiment, transgenic animals having gene knockouts are those in whichthe target gene has been rendered nonfunctional by an insertion targetedto the gene to be rendered non-functional by homologous recombination.As used herein, the term “transgenic” includes any transgenic technologyfamiliar to those in the art which can produce an organism carrying anintroduced transgene or one in which an endogenous gene has beenrendered non-functional or “knocked out.”

Detection of pH Using Fluorescent Indicator Proteins

In another embodiment, the invention provides a method for determiningthe pH of a sample by contacting the sample with an indicator includinga first fluorescent protein moiety whose emission intensity changes aspH varies between pH 5 and 10, exciting the indicator, and thendetermining the intensity of light emitted by the first fluorescentprotein moiety at a first wavelength. The emission intensity of thefirst fluorescent protein moiety indicates the pH of the sample.

The fluorescent protein moiety can be a functional engineered proteinsubstantially identical to the amino acid sequence of Aequorea greenfluorescence protein (SEQ ID NO:2). Preferred green fluorescenceproteins include those having a functional engineered fluorescentprotein that includes one of the following sets-of substitutions in theamino acid sequence of the Aequorea green fluorescent protein (SEQ IDNO:2):

S65G/S72A/T203Y/H231L

S65G/V68L/Q69K/S72A/T203Y/H231L, or

K26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L.

Other preferred green fluorescence proteins include those having afunctional engineered fluorescent protein that includes H148G or H148Qsubstitutions in the Aequorea green fluorescent protein. These proteinsinclude the YFP-H148G (SEQ ID NO:7) and YFP-H148Q (SEQ ID NO:8) proteinsdescribed above.

The sample in which pH is to be measured can be a biological sample,e.g., a biological tissue such as an extracellular matrix, blood orlymphatic tissue, or a cell. The method is particularly suitable formeasuring pH in a specific region of the cell, e.g., the cytosol, or anorganellar space such as the inner mitochondrial matrix, the lumen ofthe Golgi, cytosol, the endoplasmic reticulum, the chloroplast lumen,the lumen of lysosome, or the lumen of an endosome.

In some embodiments, the first fluorescent protein moiety is linked to atargeting sequence that directs the fluorescent protein to a desiredcellular compartment. Examples of targeting sequences include the aminoterminal 81 amino acids of human type II membrane-anchored proteingalactosyltransferase for directing the fluorescent indicator protein tothe Golgi and the amino terminal 12 amino acids of the presequence ofsubunit IV of cytochrome c oxidase for directing a fluorescent pHindicator protein to the mitochondrial matrix. The 12 amino acids of thepresequence of subunit IV of cytochrome c oxidase may be linked to thepH fluorescent indicator protein through a linker sequence, e.g.,Arg-Ser-Gly-Ile (SEQ ID NO:38).

In another embodiment, the invention provides a method of determiningthe pH of a region of a cell by introducing into the cell apolynucleotide encoding a polypeptide including an indicator having afirst fluorescent protein moiety whose emission intensity changes as pHvaries between 5 and 10, culturing the cell under conditions that permitexpression of the polynucleotide; exciting the indicator; anddetermining the intensity of the light emitted by the first proteinmoiety at a first wavelength. The emission intensity of the firstfluorescent protein moiety indicates the pH of the region of the cell inwhich the indicator is present.

The polynucleotide can be introduced using methods described above.Thus, the method can be used to measure intracellular pH in cellscultured in vitro, e.g., HeLa cells, or alternatively in vivo, e.g., incells of an animal carrying a transgene encoding a pH-dependentfluorescent indicator protein.

Fluorescence in the sample can be measured using a fluorometer. Ingeneral, excitation radiation, from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscause the excitation radiation to excite the sample. In response,fluorescent proteins in the sample emit radiation which has a wavelengththat is different from the excitation wavelength. Collection optics thencollect the emission from the sample. The device can include atemperature controller to maintain the sample at a specific temperaturewhile it is being scanned. If desired, a multi-axis translation stagecan be used to move a microtiter plate holding a plurality of samples inorder to position different wells to be exposed. The multi-axistranslation stage, temperature controller, auto-focusing feature, andelectronics associated with imaging and data collection can be managedby an appropriately programmed digital computer. The computer also cantransform the data collected during the assay into another format forpresentation.

Methods of performing assays on fluorescent materials are well known inthe art and are described in, e.g., Lakowicz, J. R., Principles ofFluorescence Spectroscopy, New York:Plenum Press (1983); Herman, B.,Resonance energy transfer microscopy, in: Fluorescence Microscopy ofLiving Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed.Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989),pp.219-243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park:Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.

The pH can be analyzed on cells in vivo, or from samples derived fromcells transfected with polynucleotides or proteins expressing the pHindicator proteins. Because fluorescent pH indicator proteins can beexpressed recombinantly inside a cell, the pH in an intracellularregion, e.g., an organelle, or an extracellular region of an organismcan be determined simply by determining changes in fluorescence.

Fluorescent protein pH sensors may vary in their respective pK_(a), andthe differences in pK_(a) can be used to select the most suitablefluorescent protein sensor for a particular application. In general, asensor protein should be used whose pK_(a) is close to the pH of thesample to be measured. Preferably the pK_(a) is within 1.5 pH unit ofthe sample. More preferably the pK_(a) is within 1 pH unit, and stillmore preferably the pK_(a) is within 0.5 pH unit of the sample.

Thus, a fluorescent protein pH sensor having a pKa of about 7. 1, e.g.,the EYFP mutant described below, is preferred for determining the pH ofcytosolic, Golgi, and mitochondrial matrix pH areas of a cell. TheYFP-H148G, YFP-H148Q, EYFP-H148G and EYFP-H148Q mutants are well-suitedfor measuring the pH of alkaline environments, e.g., mitochondrialmatrix, as they have a pKa of 7.5 and 8.0, respectively.

For more acidic organelles, a fluorescence sensor protein having a lowerpK_(a), e.g., a pK_(a) of about 6.1, is preferred.

To minimize artefactually low fluorescence measurements that occur dueto cell movement or focusing, the fluorescence of a fluorescent proteinpH sensor can be compared to the fluorescence of a second sensor, e.g.,a second fluorescent protein pH sensor, that is also present in themeasured sample. The second fluorescent protein pH sensor should have anemission spectra distinct from the first fluorescent protein pH sensorso that the emission spectra of the two sensors can be distinguished.Because experimental conditions such as focusing and cell movement willaffect fluorescence of the second sensor as well as the first sensor,comparing the relative fluorescence of the two sensors allows for thenormalization of fluorescence.

A convenient method of comparing the samples is to compute the ratio ofthe fluorescence of the first fluorescent protein pH sensor to that ofthe second fluorescent protein pH sensor.

Kits

The materials and components described for use in the methods of theinvention are ideally suited for the preparation of a kit. Such a kitmay comprise a carrier means being compartmentalized to receive one ormore container means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the container means may comprise apolynucleotide encoding a fluorescent protein pH sensor. A secondcontainer may further comprise fluorescent protein pH sensor. Theconstituents may be present in liquid or lyophilized form, as desired.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto limit the scope of the invention described in the claims.

EXAMPLE 1 Construction of Fluorescent Protein pH Sensors

Fluorescent protein pH sensors were constructed by engineeringsite-specific mutations in polynucleotides encoding forms of theAequorea victoria green fluorescent protein (GFP). The starting GFPvariant was the polynucleotide encoding the GFP variant EGFP (forenhanced green fluorescent protein). The EGFP variant had the amino acidsubstitutions F64L/S65T/H231L relative to the wild-type Aequoreavictoria GFP sequence.

The ECFP (enhanced cyan fluorescent protein) mutant was constructed byaltering the EGFP polynucleotide sequence so that it encoded a proteinhaving the amino acid substitutionsK26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L relative to wild-typeGFP amino acid sequence. A second variant, named EYFP (enhanced yellowfluorescent protein) was constructed by altering the EGFP polynucleotideto encode a protein having the amino acid substitutionsS65G/S72A/T203Y/H231L relative to the amino acid sequence of GFP. Athird variant, named EYFP-V68L-Q69K, was constructed by altering theEGFP polynucleotide to encode a protein having the amino acidsubstitutions S65G/V68L/Q69K/S72A/T203Y/H231L relative to the amino acidsequence of GFP.

A HindIII site and Kozak consensus sequence (GCCACCATG) was introducedat the 5′ end of the polynucleotide encoding the GFP variants, and anEcoR1 site was added at the 3′ end of the gene of each indicator, andthe fragments were ultimately ligated into the HindIII/EcoR1 sites ofthe mammalian expression vector pcDNA3 (Invitrogen). EGFP and EYFPmutant proteins with no targeting signals were used as indicators of pHin the cytosol or nucleus.

To construct fluorescent protein pH sensors to use as pH indicators inthe Golgi, polynucleotides encoding the 81 N-terminal amino acids of thetype II membrane-anchored protein galactosyltransferase(GT:UDP-galactose-β,1,4-galactosyltransferase. EC 2.4.1.22) ligated topolynucleotides encoding EGFP, ECFP, or EYFP. The polynucleotidesencoding the resulting proteins were named GT-EGFP, GT-ECFP, andGT-EYFP, respectively.

Mitochondrial matrix fluorescent protein pH sensors were constructed byattaching polynucleotides encoding 12 amino acids at the amino terminusof the presequence of subunit IV of cytochrome c oxidase (Hurt et al,EMBO J. 4:2061-68 (1985) to a polynucleotide encoding the amino acidsequence Arg-Ser-Gly-Ile (SEQ ID NO:38), which in turn was ligated topolynucleotides encoding ECFP or EYFP. These constructs were labeledECFP-mito or EYFP-mito.

The constructs used to examine intracellular pH are summarized in FIG.1.

EXAMPLE 2 pH Titration of Fluorescent Sensor Proteins in Vitro

The pH sensitivity of the fluorescence of the proteins ECFP, EGFP, EYFP,GT-EGFP, and GT-EYFP was first examined.

Absorbance spectra were obtained in a Cary 3E spectrophotometer(Varian). For pH titration, a monochromator-equipped fluorometer (SpexIndustries, NJ) and a 96-well microplate fluorometer (CambridgeTechnology) were used. In the latter case the filters used forexcitation were 482 10 (460 18 for ECFP) and for emission were 532 14.Filters were named as the center wavelength the half-bandwidth, both innm. The solutions for cuvette titration contained 125 mM KCl, 20 mMNaCl, 0.5 mM CaCl₂, 0.5 mM MgCl₂, and 25 mM of one of the followingbuffers—acetate, Mes, Mops, Hepes, bicine, and Tris.

EYFP showed an acidification-dependent decrease in the absorbance peakat 514 nm and a concomitant increase in absorbance at 390 nm (FIG. 2A).The fluorescence emission (527-nm peak) and excitation spectra decreasedwith decreasing pH, but the fluorescence excitation spectrum showed nocompensating increase at 390 nm. Therefore, the species absorbing at 390nm was nonfluorescent. The apparent pKa (pK′a) of EYFP was 7.1 with aHill coefficient (n) of 1.1 (FIG. 2B).

EGFP fluorescence also was quenched with decreasing pH. The pK′a of EGFPwas 6.15, and n was 0.7.

The change in fluorescence of ECFP (Tyr66→Trp in the chromophore) withpH was smaller than that of EGFP or EYFP (pK′a 6.4, n, 0.6) (FIG. 2B).The fluorescence change was reversible in the pH range 5-8.5 for allthree proteins, which covers the pH range of most subcellularcompartments. These results demonstrate that the GFP variants EGFP,EYFP, and ECFP can be used as fluorescent protein pH sensors.

EXAMPLE 3 Measurements of pH in the Cytosol and Nucleus UsingFluorescent Protein pH Sensors

HeLa cells and AT-20 cells grown on glass coverslips were transientlylipo-transfected (Lipofectin™, GIBCO) with polynucleotide constructsencoding EYFP.

Cells were imaged between 2 and 4 days after transfection at 22° C. witha cooled charge-coupled device camera (Photometrices, Tucson, Ariz.) asdescribed in Miyawaki et al., Nature 388:882, (1997). The interferencefilters (Omega Optical and Chroma Technology, Brattleboro, Vt.) used forexcitation and emission were 440 ECFP; 480 535 EGFP or EYFP. Thedichroic mirrors were 455 DCLP for ECFP and 505 DCLP for EGFP or EYFP.Regions of interest were selected manually, and pixel intensities werespatially averaged after background subtraction. A binning of 2 was usedto improve signal/noise and minimize photodamage and photoisomerizationof EYFP. High KCl buffer plus 5 μM each of the ionophores nigericin(Fluka) and monensin (Calbiochem) was used for in situ titrations inliving cells. Cells were loaded with cytosolic pH indicators byincubation with 3 μM carboxy-SNARF/AM or BCECF/AM (Molecular Probes) for45 minutes, then washed for 30 minutes, all at 22° C.

Fluorescence of HeLa cells transfected with the gene encoding EYFP wasdiffusely distributed in the cytosol and nucleus. This was expected fora protein of the size of GFP (27 kDa), which is small enough to passthrough nuclear pores.

The fluorescence observed with EYFP was reversible. Perfusion with NH₄Clcaused an increase in fluorescence (rise in pH), which reversed uponwashing out the NH₄Cl. Conversely, perfusion of lactate, which lowerspH, induced a decrease in fluorescence. The decrease in fluorescence wasalso reversible on wash-out.

Calibration of fluorescence intensity with pH in situ was accomplishedwith a mix of the alkali cation/H+ ionophores nigericin and monensin inbath solutions of defined pH and high K+. Fluorescence equilibratedwithin 1-4 minutes after each exchange of solution. These resultsdemonstrate that EYFP, when present intracellularly, can report pH inthe physiological range.

EXAMPLE 4 Measurement of pH in the Mitochondrial Matrix UsingFluorescent Protein pH Sensors

To measure pH in the mitochondrial matrix using mutant GFP sensorproteins, HeLa cells and neonatal rat cardiomyocytes were transfectedwith the fluorescent protein pH sensor EYFP-mito. A Bio-Rad MRC-1000confocal microscope was used for analysis of the targeted protein.Microscopy analysis revealed that the transfected cells showed afluorescence pattern indistinguishable from that of the conventionalmitochondrial dye rhodamine 123.

In situ pH titration was performed with nigericin/monensin as describedin Example 3. Subsequent addition of the protonophore carbonylcyanidem-chlorophenylhydrazone (CCCP) did not change the fluorescence intensityof the cells. This demonstrates that the nigericin/monensin treatmenteffectively collapsed the pH gradient (ΔpH) in the mitochondria.

The estimated pHm was 7.98 0.07 in HeLa cells (n=17 cells, from sixexperiments). Similar pH values were obtained in a HeLa cell line stablyexpressing EYFP-mito. Resting pH did not change by superfusion of cellswith medium 10 mM glucose, which would provide cells with an oxidizablesubstrate, but 10 mM lactate plus 1 mM pyruvate caused an acidification,which reversed on washout. This can be accounted for by diffusion ofprotonated acid or by cotransport of pyruvate⁻/H⁺ through the innermitochondrial membrane. The protonophore CCCP rapidly induced anacidification of mitochondria to about pH 7.

EXAMPLE 5 Measurement of pH in the Golgi Lumen Using Fluorescent ProteinpH Sensors

The type II membrane-anchored protein galactosyltransferase(GT:UDP-galactose-β,1,4-galactosyltransferase. EC 2.4.1.22) has beenused as a marker of the trans cisternae of the Golgi apparatus (Roth etal., J. Cell Biol. 93:223-29, (1982)). Accordingly, polynucleotideconstructs encoding portions of the GT protein fused to the mutant GFPproteins were constructed as described in Example 1 in order to use theGT sequence to target the fluorescent protein pH sensor to theendoplasmic reticulum.

The pH of the Golgi lumen was measured by transfecting HeLa or AT-20cells with the constructs GT-ECFP, GT-EGFP, or GT-EYFP. Brightjuxtanuclear fluorescence was observed, with little increase in diffusestaining above autofluorescnce in most cells.

The fluorescence pattern was examined further in double-labelingexperiments using rabbit polyclonal α-mannosidase II ((α-manII)antibody. Double labeling fluorescence was performed as described byMcCaffery et al., Methods Enzymol. 257:259-279 (1995). The α-manIIantibody was prepared as described in Velasco et al., J. Cell Biol.122:39-51 (1993). In the double-staining experiments, it was observedthat labeling of the medial trans-Golgi marker α-manII overlapped withGT-EYFP fluorescence.

α-manII was also fused with ECFP, and the pattern of fluorescenceobtained upon transfection of the gene was indistinguishable from thatof GT-EYFP by light microcopy.

To identify the subcellular localization of GT-EYFP at higherresolution, immunogold electron microscopy was performed on ultra-thincryosections by using antibodies against GFP. Immunogold labeling ofultra-thin sections was performed as described by McCaffery et al.,supra, using rabbit polyclonal anti-GFP antibody or a monoclonalanti-TGN38 antibody.

In double-labeling experiments, GT-EYFP was found in the medial andtrans Golgi, although endogenous GT is present in trans Golgi membranes.The difference in localization may occur as a result of overexpressionof the GT-EYFP protein.

When protein TGN38 was used as a trans-Golgi network (TGN) marker, itsimmunogold localization pattern was found to overlap with that ofGT-EYFP in the medial/trans-Golgi membranes. The localization datademonstrate that GT-EYFP labels the medial/trans Golgi. Thus, GT-EYFPcan be used to identify the pH of this organelle.

The pH titration of GT-EYFP fluorescence in the Golgi region of thecells after treatment with nigericin/monensin was in good agreement withthat of EYFP in vitro (see Example 2). Resting pH in HeLa cells was onaverage 6.58 (range 6.4-6.81, n=30 cells, 9 experiments). These resultsalso demonstrate that neither fusion with GT nor the composition of theGolgi lumen affects the pH sensitivity of EYFP. Thus, Golgi-targetedEYFP can be used as a local pH indicator.

The effect of various treatments on the pH of the Golgi was nextexamined using Golgi-targeted EYFP.

The pH gradient across the Golgi membrane is maintained by theelectrogenic ATP-dependent H⁺ pump (V-ATPase). The V-ATPase generates aΔpH (acidic inside) and Δψ(positive inside), which opposes further H⁺transport. The movement of counter-ions, Cl⁻ in (or K⁺ out), with H⁺uptake would shunt the Δψ, allowing a larger ΔpH to be generated. Thesemechanisms were investigated in intact single HeLa cells transfectedwith GT-EYFP.

The macrolide antibiotic bafilomycin A1 has been shown to be a potentinhibitor of vacuolar type H⁺ ATPases (V type). In Hela cells expressingGT-EYFP, bafilomycin A1 (0.2 μM) increased pH_(G) by about 0.6 units, topH 7.16 (range 7.02-7.37, n=12 cells. This suggests that the H⁺ pumpcompensates for a positive H+ efflux or leak. The initial rate of Golgialkalinization by bafilomycin A1 was 0.52 pH units per minute (range0.3-0.77, n=12 cells), faster than that reported for other acidiccompartments such as macrophage phagosomes (0.09 pH/min). Similarresults regarding resting pHG and alkalinization by bafilomycin A1 wereobtained when HeLa cells were transfected with GT-EGFP. Calibration ofGT-EGFP in situ also mirrored its in vitro titration (FIG. 1B). Thus,both EGFP and EYFP are suitable Golgi pH indicators.

EXAMPLE 6 Measuring Intercellular pH with Two Fluorescent ProteinSensors

Quantitative measurements of fluorescence with nonratiometric indicatorscan suffer from artifacts as a result of cell movement or focusing. Tocorrect for these effects, the cyan-emitting mutant GT-ECFP wasco-transfected into cells along with GT-EYFP. ECFP has excitation andemission peaks that can be separated from those of EYFP by appropriatefilters. In addition, ECFP is less pH-sensitive than EYFP (see FIG. 2B).

FIG. 3A demonstrates that the fluorescence of ECFP changed less thanthat of EYFP during the course of the experiment. Although the ratio ofEYFP to ECFP emission varied between cells, probably reflecting adifferent concentration of GT-EYFP and GT-ECFP in the Golgi lumen, itchanged with pH as expected (FIG. 3B). Bafilomcin A1 raised theGT-EYFP/GT-ECFP emission ratio, i.e, it raised pH_(G).

EXAMPLE 7 Construction of YFP THR H148G and YFP H1480 Mutants

The YFP H148G mutant was prepared using as a template a nucleic acidencoding the YFP mutation 10c, which includes the mutationsS65G/V68L/S72A/Q80R/T203Y and is described in Ormö et al., Science273:1392-95 (1997). The YFP H148G mutant,was constructed using thePCR-based QUIKCHANGE™ Site-Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.) following the manufacturer's instructions. The YFP H148Qmutation was similarly constructed from a nucleic acid encoding the 10Cmutation.

The pKa of the YFP H148G mutant was found to be 8.0, while the YFP H148Qmutant was found to have a pKa of 7.5.

EXAMPLE 8 Expression of mito-YFP H148G in the Mitochondrial Matrix at apH Range of 7.0 to 8.4

The high pKa of the mutant YFP H148G allows it the to be used for theprecise measurement of mitochondrial matrix pH both in cells at rest andin cells subject to manipulations that decrease mitochondrial pH.

This was demonstrated directly by transfecting a nucleic acid encodingmito-YFP H148G into HeLa cells using the procedures described in Example4. YFP H148G expression was monitored by observing fluorescence overtime. Mitochondrial pH was also monitored by pH titration as describedin Example 3 using nigericin and monensin.

FIG. 4 shows that HeLa cells transfected with YFP H148G in themitochondrial matrix were fluorescent at an initial pH of 8.0 to 8.1(where measurements began at t≈0 seconds). 5 μM CCCp was added at aboutt≈300 seconds. Although addition of 5 μm CCCP rapidly lowered the pH to7.0, fluorescence of mito-YFP H148G was still detectable. Then acalibration was performed by perfusing the cells with extracellularmedium of pH 7, 7.5, 8, and 8.35 containing the ionophores nigericinplus monensin to equilibrate mitochondrial pH and extracellular pH.Fluorescence in mitochondria increased stepwise with each change ofextracellular pH.

Fluorescence was also examined, and pH measured, in primary cultures ofchick skeletal myotubes transfected with the mito-YFP H148G mutant. FIG.5 demonstrates that fluorescence was detectable in the mitochondrialmatrix of chicken skeletal myotubes, which had a pH of 8.0-8.1 (t≈0).Fluorescence was still detectable following addition of 25 μM forskolin,which did not affect the pH, and after addition of 2 μM CCCP at t≈750seconds, although CCCP caused the pH to rapidly drop to 6.9 at t≈1400seconds. Thereafter fluorescence continued to be observed duringcalibration at ph 6.9, 7.6 and 8.0.

These results demonstrate mito-YFP H148G fluorescence is detectable inthe mitochondrial matrix over the pH range of 7.0 to 8.4 in bothestablished cell lines (HeLa cells) and primary cultures (chick skeletalmyotubes).

EXAMPLE 9 Expression of mito-YFP H1480 in Response to PH Changes

The YFP H148Q mutant has a pKa of about 7.4, which is intermediatebetween the pKa of EYFP and YFP mutant H148G. To demonstrate that YFPH148Q can also be used to measure mitochondrial matrix pH, a nucleicacid encoding mito-YFP-H148Q was transfected into HeLa cells.Fluorescence was measured over time (beginning at t≈0), includingfollowing the addition of 10 μM nigericin in high KCL titration bufferat t≈500 seconds.

FIG. 6 reveals the effect of changing mitochondrial pH to 6.9, 7.5, 8,and 8.4 with the ionophore nigericin on fluorescence intensity.Fluorescence decreased to about 175 units at t=1000 seconds by additionof nigericin, which lowered the pH to about 6.9. Fluorescence thenreturned stepwise to 400 units with each change of the extracellularmedium. These results demonstrate that the fluorescence of the mito-YFPH148Q mutant can be used to measure the pH of the inner mitochondrialmatrix.

EXAMPLE 10 Measuring Intercellular pH by Coexpression of YFP H148G and aSecond PH-Insensitive Sensor

As is discussed above in Example 6, for quantitative measurements it isdesirable to compute the fluorescence of the sensor used to measure pHwith the fluorescence of a second sensor molecule whose fluorescencedoes not change over the pH range being tested. A ratiometricmeasurement is useful to correct for movement or focusing artifacts thatmay occur during live cell imaging experiments.

To identify a GFP sensor protein suitable for use as a reference proteinfor measuring mitochondrial matrix pH, the GFP mutant T203I wasexpressed in the mitochondria of HeLa cells. The GFP T203I mutant can beexcited with light of 400 nm, which does not appreciably excite the pHsensitive YFP mutants.

Fluorescence of HeLa cells transfected with the GFP T203I mutant wasmonitored for about 400 seconds using an excitation ratio of 480 nm/400nm. 10 μM CCCP was then added to the cells, and fluorescence wasmonitored for an additional 250 seconds. Addition of CCCP did not affectfluorescence. In control experiments, it was observed that addition ofCCCP corresponded to a drop in pH of about 1 unit. Thus, the GFP T203Imutant is suitable for use as a reference, pH-insensitive mutant.

HeLa cells were then transfected with the GFP T203I mutant and YFPH148G. FIG. 7 shows the change of mitochondrial pH with oligomycin andthe uncoupler CCCP as the ratio of YFP H148G emission and GFP T203Iemission, with excitation of 490 and 400 nm, respectively.

EXAMPLE 11 Structural Characterization of YFP T203Y/S65G/V68L/S72A/H148G

The green fluorescent protein (GFP) from the jellyfish Aequorea victoriahas been used extensively in molecular biology as a fluorescent label.The structures of WT GFP (Yang et al., Nature Biotech. 14:1246-51, 1996;Brejc et al., Proc. Natl. Acad. Sci. USA. 94:2306-11, 1997) and thevariant S65T were determined in 1996 (Ormö et al., Science 273:1392-95,1996).

A large number of mutants have been identified that exhibit broadlyvarying absorption and emission maxima (Heim et al. above; Heim et al.,Curr. Biol. 6: 178-82, 1996). The yellow fluorescent protein (YFP)mutant is of particular interest since its spectrum is shifted enough torender it readily distinguishable from the spectrum of Cyan FluorescentProtein (CFP) for FRET measurements (Tsien, Ann. Rev. Biochem. 67:509,1998; Miawaki et al., Nature 388:882-87, 1997). WT GFP exhibits twoabsorption maxima, where the major band absorbs at 398 nm and the minorband at 475 nm (Morise et al., Biochemistry 13:2656-62, 1974).Excitation of either of these bands leads to emission of green lightwith a maximum between 504 and 508 nm (FIG. 8). Before a structure wasavailable, GFP variants with altered spectral characteristics wereidentified by random mutagenesis. Some of these mutants, such as Y66Hand Y66W (Tsien, Ann. Rev. Biochem. 67:509, 1998, Heim et al., Proc.Natl. Acad. Sci. (USA) 91:12501-04) result in blue-shifted absorbanceand emission maxima. Others focus on changes in the immediateenvironment of the chromophore π system, such as S65T (Heim et al.,Nature 373:.663-64, 1995) and T203I (Heim et al., Proc. Natl. Acad. Sci.(USA) 91:12501-04, 1994). At physiological pH, S65T exhibits only onemajor absorption band at 489 nm, red-shifted by 14 nm from WT GFP, andis almost six times brighter (Heim et al., Nature 373: 663-64, 1995).Yet, the emission spectrum is shifted by only 3 nm to 511 nm, and socannot easily be distinguished from the wild-type emission. Randommutagenesis techniques produced only one further red-shifted variant,S65T/M153A/K238E, which increases the excitation and emissionwavelengths of S65T by 15 and 3 nm respectively (Heim et al., Curr.Biol. 6: 178-82, 1996). Here is described crystal structures of thefirst set of GFP variants rationally designed based on the x-raystructure of GFP S65T (Ormö et al., Science 273:1392-95, 1996). Thesevariants, termed YFPs (Yellow Fluorescent Proteins), exhibit the longestwavelength emissions of all GFPs generated by mutagenesis (FIG. 8). TheYFPs fluoresce around 528 nm, red-shifted by 16 nm as compared to S65Tand are easily distinguishable from S65T on a fluorescence microscope.

The specific YFP investigated is the quadruple-mutantT203Y/S65G/V68L/S72A, where the substitution T203Y was introduced basedon the structural considerations detailed below and is believedresponsible for the red-shift. The other three mutations have been shownto improve its brightness in live cells (Cormack et al., Gene 173:33,1996). The T203Y mutation would have been difficult to identify byrandom mutagenesis since this amino acid substitution requires threesubstitutions at the nucleotide level. Since Thr203 is positioned closeto the chromophore, it was postulated that its replacement with atyrosine would result in π-stacking interactions between the chromophoreand the highly polarizable phenol (Ormö et al., Science 273:1392-95,1996), leading to red-shifted spectral properties. The structure of S65Tsuggested that an aromatic amino acid introduced in position 203 wouldextend into the water-filled cavity adjacent to the chromophore (Ormö etal., Science 273:1392-95, 1996). Replacement of Thr203 with any of thearomatic amino acids His, Trp, Tyr, or Phe was found to lead to thedesired spectral shifts (Ormö et al., Science 273:1392-95, 1996, Dicksonet al., Nature 388:355-58, 1997). The most dramatic red-shift wasobserved for the T203Y substitution, therefore this variant has beentermed YFP.

In order to determine the role of His148 in modulating the pKa of thechromophore or its spectral properties, an additional mutation, H148G,was introduced into the YFP background. The x-ray structures of YFP andYFP H148G were analyzed in order to better correlate structural changeswith spectral properties. GFP variants were prepared as described inExample 6, above. This template incorporates the mutationsT203Y/S65G/V68L/S72A, as well as the ubiquitous Q80R substitution thatwas accidentally introduced into the gfp cDNA early on (Ormö et al.,Science 273:1392-95, 1996; Chalfie et al., Science 263:802-05, 1994).All GFP variants were expressed and purified as described (Ormö et al.,Science 273:1392-95, 1996).

Structural Determination of YFP H148G

YFP H148G was concentrated to 12 mg/ml in 20 mM HEPES pH 7.9. Rod-shapedcrystals with approximate dimensions of 1.8×0.08×0.04 mm were grown inhanging drops containing 2 μl protein and 2 μl mother liquor at 4° C.within four days. The mother liquor contained 16% PEG 4000, 50 mM sodiumacetate pH 4.6, and 50 mM ammonium acetate. X-ray diffraction data werecollected from a single crystal at room temperature using a Xuong-Hamlinarea detector (Hamlin, Methods. Enzymol. 114:416-52, 1985). Data werecollected in space group P2₁2₁2₁ to 99% completeness at 2.6 Åresolution, and reduced using the supplied software (Howard et al.,Methods Enzymol. 114:452-71, 1985). Unit cell parameters were a=52.0,b=62.7, and c=69.9. The GFP S65T coordinate file (Ormö et al., Science273:1392-95, 1996) which served as a model for phasing was edited toreflect the mutations, with the introduced residues Tyr203 and Leu68initially modeled as alanines to prevent model bias. A model for theanionic chromophore was obtained by semi-empirical molecular orbitalcalculations using AM1 in the program SPARTAN version 4.1 (WavefunctionInc., Irvine, Calif.). The minimized structure, which was planar,compared very favorably with a related small molecule crystallographicstructure (Tinant et al., Cryst. Struct. Comm. 9:671-74, 1980), and alsowith the model used during refinement of GFP S65T, where a simplermodeling program had been employed (Ormö et al., Science 273:1392-95,1996).

Using the program TNT (Tronrud et al., Acta Crystallogr. Sect. A 43:489,1987), rigid body refinement was carried out to position the isomorphousmodel in the unit cell of YFP H148G. Initial positional refinement wascarried out using the data to 4.0 Å, then to 3.5, 3.0, and finally to2.6 Å. Electron density maps (2Fo−Fc and Fo−Fc) were inspected using O(Tronrud et el., above), and solvent molecules were added if consistentwith Fo−Fc features, and only when in proximity of hydrogen bondpartners. B-factors were refined using a strong correlation betweenneighboring atoms due to the relatively low resolution. Since noB-factor library is available for the chromophore itself, the B-factorsof all chromophore atoms were set to the values obtained in the 1.9 Åstructure of GFP S65T (Ormö et al., Science 273:1392-95, 1996), and thenrefined as a group, with identical shifts for the grouped atoms.Structure Determination of YFP.

YFP was concentrated to 10 mg/ml in 50 mM HEPES pH 7.5. After 2 weekscrystals grew to a size of 0.03×0.12×0.8 mm at 15° C. in hanging dropscontaining 5=1 protein and 5=1 well solution, which contained 2.2 Msodium/potassium phosphate pH 6.9. These crystals belong to space groupP2₁2₁2 and have the unit cell dimensions a=77. 1, b=117.4, w and c=62.7.X-ray diffraction data were collected on two isomorphic crystals at roomtemperature using an Raxis-IV imaging plate mounted on a Rigaku RUH3rotating anode generator equipped with mirrors. The data were processedwith Denzo and scaled using ScalePack (Otwinowski et al., MethodsEnzymol. 276:307-26, 1997). The YFP structure was solved by molecularreplacement using the program AMoRe (Navaza, Acta Crystallogr.A50:157-63, 1994), with the 1.9 Å GFP S65T coordinate file as the searchmodel (Ormö et al., Science 273:1392-95, 1996). Two solutions wereidentified, consistent with two molecules per asymmetric unit.

For refinement, the 2.6 Å structure of YFP H148G was chosen as theinitial model, which was edited to reflect the mutations present in YFP.To avoid model bias, the occupancies of the Tyr203 side chain atoms andall chromophore atoms were set to zero during the first several roundsof refinement. Constrained NCS averaging over the A and B chains in theasymmetric unit was applied, initial refinement was carried out to 3.5 Åonly, and the electron density maps (2Fo−Fc and Fo−Fc) were averaged.These maps were then inspected, and the model adjusted using O (Jones etal., Acta Crystallogr. Sect. A 47:110, 1991), followed by additionalpositional refinement to 2.5 Å. Chromophore and Tyr203 densities werevery clear, and both were planar. The model was edited to include thesegroups in refinement, and solvent molecules were added whereappropriate. B-factors were refined using a strong correlation betweenneighboring atoms due to the relatively low resolution.

Comparison of the Structure of YFP and YFP H148G

YFP crystallized in 2.2 M Na/K phosphate at pH 6.9 in spacegroup P2₁2₁2,with 2 molecules per asymmetric unit (chains A and B). The GFP S65Tstructure was used as a search model for molecular replacement against a3.0 Å dataset using the program AMoRe (Navaza, Acta Crystallogr.A50:157-63, 1994), and the structure was refined. Later, the refinedstructure of YFP H148G (see below) was used for phasing and refinementagainst a 2.5 Å dataset. Even though the introduced Tyr203 and thechromophore itself were not modeled during early cycles of refinement,clear electron density for a planar chromophore and a stacked Tyr203phenol was immediately apparent. Non-crystallographic symmetry (NCS)constraints were employed throughout refinement of the model using TNT,and maps were averaged. At the end of refinement, non-averaged maps forthe A- and B-chain in the asymmetric unit were calculated and comparedto each other. No obvious features were identifiable that would suggestsignificant differences between the two chains. A test run of refinementwithout any NCS constraints confirmed that the differences would besmaller than the rms error of a 2.5 Å structure. Therefore, the NCSconstraints were not relaxed or eliminated. Data collection and atomicmodel statistics are shown in Table 33. The final R-factor of the YFPmodel was 19.2% for all data between 20 and 2.5 Å resolution.

TABLE 33 Data collection and atomic model statistics of YFP and YFPH148G YFP YFP H148G Total observations 53,039 29,904 Unique reflections18,916 7,373 Completeness^(a) 92% 99% Completeness (shell^(b)) 94% 97%Number of crystals 2 1 ^(R)merge^(c) (%) 8.0% 6.5% Resolution 2.5 Å 2.6Å Atomic model statistics: Spacegroup P2₁2₁2 P2₁2₁2₁ Molecules perasymm, unit 2 1 Crystallographic R-factor 0.192 0.159 Protein atoms1,810 1,810 Solvent atoms per asymmm, unit 130 30 Bond length deviations(Å) 0.013 0.012 Bond angle deviations (°) 1.76 2.07 Thermal parameterrestraints (Å²) 4.53 3.82 ^(a)Completeness is the ratio of the number ofobserved I > 0 divided by the theoretically possible number ofintensities. ^(b)Shell is the highest resolution shell (2.56 to 2.50 Åfor YFP, and 2.80 to 2.60 Å for YFP H148G) ^(c)R_(merge) = Σ |I_(hkl) −<I>| / Σ < I > where < I > = average of individual measurements ofI_(hkl)

The refined YFP structure clearly shows that the overall fold isundisturbed, with an rms deviation from the GFP S65T structure of 0.36 Åfor α-carbons. Three larger contact areas with adjacent molecules wereidentified. The largest of these covers about 722 Å2 of one monomersurface, includes a series of hydrophobic residues consisting of Ala206,Phe223, and Leu221, and also a number of hydrophilic contacts. Thisinterface is essentially identical to the dimer interface for WT GFPdescribed by Yang et al., Nature Biotech. 14:1246-51, 1996. High saltconditions during crystallization experiments appear to favordimerization, as has been suggested previously (Palm et al., Nat.Struct. Biol. 4:361-65, 1997).

YFP H148G crystallized as a monomer in the presence of polyethyleneglycol and acetate at pH 4.6 in spacegroup P2₁2₁2, isomorphous to S65T(Ormö et al., Science 273:1392-95, 1996) and the blue-emission variantBFP (Wachter et al., Biochemistry 36:9759-65, 1997). Molecularreplacement using the S65T structure for phasing and refinement gave afinal model with an R-factor of 15.9% for all data between 24.0 and 2.6Å (Table 33). As with YFP, electron density for a stacked phenol wasclearly visible even before the Tyr203 ring was modeled. The rmsdeviation between YFP H148G and S65T a-carbons is 0.31 Å, and thedeviation between YFP H148G and YFP a-carbons is 0.35 Å. The b-strandsof the two YFP variants overlay closely in all areas except around theC_(α) of residue 148 where a movement of 1.1 Å is observed. Thismovement has not been observed in other pH 4.6 structures grown undersimilar conditions and crystallizing in the same space group, such asthe BFP structure (Wachter et al., Biochemistry 36:9759-65, 1997).Residue 148 and adjacent residues are not involved in crystal contacts,further indicating that the observed movement is due to the H148Gsubstitution, not crystallization conditions.

π-Stacking of the Introduced Phenol

Electron densities of the chromophore and the phenol ring of Tyr203appeared to be completely planar before the atoms for these groups wereadded to the model. When the tyrosine side chain was first introducedinto the model, it was modelled as co-planar to the chromophore.Refinement consistently rotated the phenol ring by 12 with respect tothe chromophore plane in both YFP and YFP H148G. FIG. 9 shows theelectron density of the refined YFP chromophore structure together withthe phenol ring of Tyr203. The distance of the closest approach betweenatoms of the two interacting rings is 3.3 to 3.4 Å, and occurs at thatedge of the chromophore plane that is opposite the exo-methylene bond(FIG. 9). It appears that the phenol tilts towards this area of thechromophore since it is more open, with fewer atoms to clash withsterically.

The distance of largest separation between the rings is 3.5 to 3.8 Å,and occurs at the opposite edge, where steric clash with theexo-methylene carbon could occur. This range of plane-to-plane distancesis typical for face-to-face π to π stacking interactions found inproteins, and consistent with interaction energy calculations that showa potential energy minimum for two horizontally stacked benzenemolecules with a vertical separation of 3.3 Å (Burley et al., J. Am.Chem. Soc. 108, 7995-8001, 1986). A recent analysis of proteinstructures has led to the conclusion that aromatic ring interactions inan off-centered parallel orientation have an energetically favorable,stabilizing effect, and in fact are the preferred interactions(McGaughey et al., J. Biol. Chem. 273, 15458-63, 1998).

Positional Shift of the Chromophore

The entire chromophore ring system of YFP has moved out towards theprotein surface by about 0.9 Å when compared to S65T or WT GFP. Thechromophore of YFP H148G has moved in the same direction but to a lesserextent, about 0.5 Å. Overlay of all α-carbons shows that this shift isvery much a local effect, only involving residues 65 to 68. The overlaysuggests that this shift may be due to the compensating effects of theV68L and S65G substitutions. The Leu68 Cδ1 occupies the same space asthe original Va168 Cγ1, whereas the Leu68 backbone is displaced so thatthe chromophore is pushed further out towards the protein surface. Aspart of the same movement, the. Cα of Gly65 is pushed into the positionof the wild-type C_(β) of Ser65. The V68L and S65G substitutions hadbeen previously found to significantly increase the brightness ofGFP-expressing cells (Cormack et al., Gene 173:33, 1996) in a WTbackground, and were suggested to either improve folding at 37° orincrease the rate of chromophore formation. It is unclear at this pointwhy the chromophore is not shifted to the same extent in the YFP and YFPH148G structures, though both of them incorporate the V68L and S65Gmutations.

Even though the imidazolinone ring of the YFPs is not in the sameposition as in WT GFP (Brejc et al., Proc. Natl. Acad. Sci. USA.94:2306-11, 1997), S65T (Ormö et al. Science 273:1392-95, 1996), andblue-fluorescent protein BFP (Wachter et al., Biochemistry 36:9759-65,1997), no electron density consistent with partially formed or unformedchromophore is observed. This indicates that the machinery to generatethe chromophore is not only intact, but more flexible than previouslythought. Apparently, the exact positions of the backbone atoms ofresidues 65 and 67 that undergo the cyclization reaction is not ascrucial as was previously suggested, based on the nearly exactsuperposition of the imidazolinone rings observed in WT GFP, S65T, andBFP (Yang et al., Nature Biotech. 14:1246-51, 1996; Brejc, K. et al.,Proc. Natl. Acad. Sci. USA 94:23Q6-2311, 1997; Ormö et al., Science273:1392-95, 1996; Palm et al., Nat. Struct. Biol. 4:361-65, 1997;Wachter et al., Biochemistry 36:9759-65, 1997).

Chromophore Spectral Properties, Charge State and Hydrogen BondingInteractions

The spectral properties of the YFPs were examined. Small aliquots ofprotein (16 mg/ml) were diluted 48-fold into 75 mM buffer (acetate,phosphate, Tris, or CHES), 140 mM NaCl, and then scanned for absorbancebetween 250 and 600 nm (Shimadzu 2101 spectrophotometer at medium scanrate and room temperature). The optical density at 514 or 512 nm wasplotted as a function of pH and computer-fitted to a titration curve(Kaleidagraph™, SynergySoftware).

Fluorescence measurements were carried out on a Hitachi F4500fluorescence spectrophotometer at a constant protein concentration ofapproximately 0.01 mg/ml, with buffer conditions identical to those ofabsorbance measurements. The excitation wavelength was set to theabsorbance maximum of the long-wave band of the particular mutant. Theemission was scanned between 500 and 600 nm, and peak emission intensitywas plotted as a function of pH and curve-fitted.

Like S65T (Kneen et al., Biophys. J. 74:1591-99, 1998), the YFPs havetwo absorbance maxima whose relative ratio is pH-dependent (FIG. 8 andTable 34). The UV absorption peaks at 392 (YFP) or 397 nm (YFP H148G)have been ascribed to the neutral chromophore, whereas the visibleabsorption peaks at 514 (YFP) or 512 nm (YFP H148G) have been ascribedto the anionic chromophore (Niwa et al., Proc. Natl. Acad. Sci. (USA)93: 13617-22, 1996). The lower energy peak exhibits clear vibrationalstructure as indicated by the pronounced shoulder at 480-490 nm, and itsmirror-image relationship with the emission band is striking (FIG. 8).These features are consistent with luminescence properties of large andrigid systems in condensed phases (Barltrop et al., Principles ofPhotochemistry, John Wiley and Sons, New York, 1978, pp. 51-52 and78-79), and may be more pronounced in the YFPs due to decreasedchromophore flexibility in the presence of the stacked phenol. Both YFPsfluoresce intensely when excited at the longer-wavelength band, withmaximum emission occurring at 528 nm (FIG. 8). Fluorescence is extremelyweak when the excitation occurs at the shorter-wavelength band (Table34), even if the experiment is carried out at a pH where this peakdominates. The chromophore pKa in the intact protein was determined tobe 7.00.(YFP and 8.02.(0.01) for YFP H148G by absorbance measurements atvarying pH. The pKa values determined by fluorescence were 6.95.(0.03)and 7.93.(The YFP pKa is remarkably similar to that ofEYFP(S65G/S72A/T203Y/H231L). All titration curves gave an excellent fitto a single pKa value.

TABLE 34 Summary of Absorption and Emission Maxima absorbance absorbanceemission^(a) emission^(a) band #1 band #2 band band WT GFP 398 475460/508 504 S65T 394 489 (weak) 511 YFP 392 514 (weak) 528 YFP-H148G 39751122  (weak) 528 ^(a)The emission band #1 results from excitation atthe absorbance peak #1, and the emission band #2 results from excitationat the absorbance peak #2.

It is likely that the charge state of the chromophore is mixed in theYFP crystals which were grown at pH 7, and which is the chromophore pKa.In YFP, His148 is directly hydrogen-bonded to the phenolic end of thechromophore. Its electron density is well-defined, suggesting that theimidazole ring does not change position when the chromophore ionizes. Itis therefore unlikely that structural rearrangements in the immediatechromophore environment occur in response to changes in chromophorecharge state. In both the YFP and YFP H148G structures, the phenolic endof the chromophore is nearly in H-bonding contact with bulk solvent viatwo ordered waters, and therefore may not be as tightly embedded in theprotein as in WT and S65T (Brejc et al., Proc. Natl. Acad. Sci. USA.94:2306-11, 1997, Ormö et al., Science 273:1392-95, 1996]. Structuralreadjustments to accommodate the anion may only affect solventmolecules.

The strong hydrogen bond to Arg96 that has been suggested to play a rolein the chemistry of backbone cyclization (Ormö et al., above) ismaintained in both structures. The carbonyl oxygen of the chromophoreimidazolinone ring interacts with two hydrogen bond donors, Arg96 andGln69 in YFP, and Arg96 and Gln94 in YFP H148G. This compares to similarinteractions with Arg96 and Gln94 in WT and S65T. The Glu222 carboxyoxygen approaches the chromophore imidazolinone ring nitrogen to within3.0 (YFP) and 3.3 Å (YFP H148G), considerably closer than in WT and S65T(4.3 and 4.0 Å, respectively). This close approach appears to be relatedto the chromophore positional shift described above. Distance andgeometry for hydrogen bonding between Glu222 and the chromophore ringnitrogen are excellent in YFP, and somewhat less optimal in YFP H148G,where the presumed H-bond makes roughly a 45° angle with the chromophoreplane. The YFP structure is the first GFP structure solved that suggestsH-bonding interactions of the heterocyclic ring nitrogen originatingfrom Tyr66. The most likely interpretation in terms of charge states isa deprotonated ring nitrogen and a protonated Glu222, rendering bothgroups neutral, however, it is clear that they share a proton.

Solvent-accessible Surface and Cavities

The mutation H148G was introduced into YFP to examine the effects ofsolvent accessibility on the fluorescent properties and the ionizationconstant of the chromophore. In all GFP structures examined to date, theβ-barrel is somewhat perturbed around the phenolic end of thechromophore. The β-strand that covers the chromophore in that areabulges out around His148, so that the backbone from residue 144 to 150is not directly hydrogen-bonded to the adjacent backbone betweenresidues 165 and 170. Rather, they are laced together by forming H-bondswith the imidazole ring of His148 (Arg168 backbone N to His148 N_(ε2) inS65T and WT GFP) and several water molecules. The phenolic end of thechromophore is located directly “behind” the ring of His148. It wasanticipated that substitution of His with Gly would open up a solventchannel to the chromophore in the absence of other structuralperturbances, or perhaps to permit the bulge to close.

The crystal structure clearly shows this anticipated solvent channel asan invagination of the protein surface with no ordered solvent moleculeswithin. Elimination of the imidazole ring in the H148G substitution onlyleads to minor structural rearrangements of protein groups. Theβ-strands do not close up to form a directly H-bonded sheet betweenresidues 144 and 150. Instead, the Cα of residue 148 has actually movedin the opposite direction by 1.1 Å, causing an even larger strandseparation between the backbones of residues 148 and 168. The side chainof Ile167 has moved by 1.1 Å towards the space previously occupied bythe imidazole ring. Nevertheless, direct solvent access to the phenolicend of the chromophore is greatly improved. Calculation of thesolvent-accessible area of the chromophore using a probe sphere ofradius of 1.4 Å (Connolly, Science 221:709-13, 1983), as implemented byUCSF MidasPlus (UCSF MidasPlus®, Computer Graphics Laboratory,University of San Francisco, Calif. 94143), shows that 22% of thechromophore surface is solvent-accessible. Only the phenolic end of thechromophore is exposed to exterior solvent, though, due to the openingin the protein wall. The phenolic oxygen of the chromophore is alsohydrogen-bonded to a water molecule that is near H-bonding distance to asurface water, though a 1.4 Å probe cannot access the chromophore viathis path. If both the solvent channel as well as this hydrogen bond areincluded, 8% of the chromophore surface is accessible to exteriorsolvent, entirely at the phenolic end, and 14% is accessible to interiorsolvent due to contact with internal cavities.

YFP H148G contains two larger interior cavities that are in contact withthe chromophore cavity and filled with some ordered waters. The cavitythat was largest in S65T has decreased in size from approximately 127 Å3(S65T) to 88 Å3 (YFP H148G), because some of the space is now filledwith the phenol of Tyr203. In YFP, this cavity is not accessible to a1.4 Å probe at all since several groups have moved into this space. Themore significant structural adjustments are C_(γ)2 of Va1224, which hasmoved by 1.4 Å, and C_(δ1) of Leu42 which has moved by 2.0 Å,essentially filling the cavity. The second larger cavity in contact withthe chromophore is nearly invariant for S65T, YFP, and YFP H148G, and isbetween 98 and 103 Å³ in size.

Solvent Accessibility to the Chromophore

YFP H148G was found to be highly fluorescent, with brightgreenish-yellow color under ordinary day light. The light-emittingproperties of the fluorophore do not appear to be changed to any extentby the introduction of a solvent channel to the chromophore, indicatingthat significant quenching does not occur.

Since the protein fold is entirely intact in YFP H148G in spite of thegeneration of an opening in the β-barrel, the H148G substitution may beespecially useful for allowing access of various small-molecule speciesto the chromophore. This substitution may be introduced into other GFPvariants with a larger cavity adjacent to the chromophore, such as S65T[7] or S65G, allowing for analyte binding studies where specificspectral shifts due to the interaction with small molecules or ions ofinterest could be monitored. The highest ionization constant of allvariants examined to date is found for the YFP H148G mutant with a pKaof 8.0. In this mutant, the chromophore is solvent exposed, consistentwith a similarly high pKa when the protein is denatured (Nageswara etal., Biophys. J. 32:630-32, 1980).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

38 1 716 DNA Aequorea victoria CDS (1)...(714) Green fluorescent protein1 atg agt aaa gga gaa gaa ctt ttc act gga gtt gtc cca att ctt gtt 48 MetSer Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15gaa tta gat ggt gat gtt aat ggg cac aaa ttt tct gtc agt gga gag 96 GluLeu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 ggtgaa ggt gat gca aca tac gga aaa ctt acc ctt aaa ttt att tgc 144 Gly GluGly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 act actgga aaa cta cct gtt cca tgg cca aca ctt gtc act act ttc 192 Thr Thr GlyLys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 tct tat ggtgtt caa tgc ttt tca aga tac cca gat cat atg aaa cgg 240 Ser Tyr Gly ValGln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 cat gac tttttc aag agt gcc atg ccc gaa ggt tat gta cag gaa aga 288 His Asp Phe PheLys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act ata ttt ttcaaa gat gac ggg aac tac aag aca cgt gct gaa gtc 336 Thr Ile Phe Phe LysAsp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttt gaa ggtgat acc ctt gtt aat aga atc gag tta aaa ggt att 384 Lys Phe Glu Gly AspThr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gat ttt aaa gaagat gga aac att ctt gga cac aaa ttg gaa tac aac 432 Asp Phe Lys Glu AspGly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 tat aac tca cacaat gta tac atc atg gca gac aaa caa aag aat gga 480 Tyr Asn Ser His AsnVal Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 atc aaa gttaac ttc aaa att aga cac aac att gaa gat gga agc gtt 528 Ile Lys Val AsnPhe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 caa cta gcagac cat tat caa caa aat act cca att ggc gat ggc cct 576 Gln Leu Ala AspHis Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctt ttacca gac aac cat tac ctg tcc aca caa tct gcc ctt tcg 624 Val Leu Leu ProAsp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 aaa gat cccaac gaa aag aga gac cac atg gtc ctt ctt gag ttt gta 672 Lys Asp Pro AsnGlu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 aca gct gctggg att aca cat ggc atg gat gaa cta tac aaa 714 Thr Ala Ala Gly Ile ThrHis Gly Met Asp Glu Leu Tyr Lys 225 230 235 ta 716 2 238 PRT Aequoreavictoria 2 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile LeuVal 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val SerGly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys PheIle Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val ThrThr Phe 50 55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His MetLys Arg 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr ValGln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr ArgAla Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile GluLeu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly HisLys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met AlaAsp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile ArgHis Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr GlnGln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp AsnHis Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn GluLys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala GlyIle Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 3 239 PRT Aequoreavictoria VARIANT (0)...(0) EGFP 3 Met Val Ser Lys Gly Glu Glu Leu PheThr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val AsnGly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr TyrGly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro ValPro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys PheSer Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys SerAla Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys AspAsp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly AspThr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys GluAsp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn SerHis Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly IleLys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 ValGln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225230 235 4 239 PRT Aequorea victoria VARIANT (0)...(0) EYFP 4 Met Val SerLys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val GluLeu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu GlyGlu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys ThrThr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Phe GlyTyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 GlnHis Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 ArgThr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu AspGly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro IleGly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser TyrGln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His MetVal Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly MetAsp Glu Leu Tyr Lys 225 230 235 5 239 PRT Aequorea victoria VARIANT(0)...(0) EYFP-V68L/Q69K 5 Met Val Ser Lys Gly Glu Glu Leu Phe Thr GlyVal Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly HisLys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly LysLeu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro TrpPro Thr Leu Val Thr Thr 50 55 60 Phe Gly Tyr Gly Leu Lys Cys Phe Ala ArgTyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala MetPro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp GlyAsn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr LeuVal Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp GlyAsn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His AsnVal Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys ValAsn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln LeuAla Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro ValLeu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 195 200 205 SerLys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 2356 239 PRT Aequorea victoria VARIANT (0)...(0) ECFP 6 Met Val Ser Lys GlyGlu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu AspGly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu GlyAsp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr GlyLys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Trp GlyVal Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His AspPhe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr IlePhe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val LysPhe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 IleAsp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145 150155 160 Gly Ile Lys Ala His Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly AspGly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln SerAla Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val LeuLeu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp GluLeu Tyr Lys 225 230 235 7 238 PRT Aequorea victoria VARIANT (0)...(0)YFP H148G 7 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile LeuVal 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val SerGly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys PheIle Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val ThrThr Phe 50 55 60 Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His MetLys Arg 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr ValGln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr ArgAla Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile GluLeu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly HisLys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser Gly Asn Val Tyr Ile Met AlaAsp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile ArgHis Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr GlnGln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp AsnHis Tyr Leu Ser Tyr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn GluLys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala GlyIle Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 8 238 PRT Aequoreavictoria VARIANT (0)...(0) YFP H148Q 8 Met Ser Lys Gly Glu Glu Leu PheThr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val AsnGly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr TyrGly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro ValPro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Gly Tyr Gly Leu Gln Cys PheAla Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 His Asp Phe Phe Lys SerAla Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys AspAsp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly AspThr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys GluAsp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn SerGln Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 IleLys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser 195200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225230 235 9 239 PRT Aequorea victoria VARIANT (0)...(0) EYFP-H148G 9 MetVal Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60Phe Gly Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 7580 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 9095 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu GluTyr 130 135 140 Asn Tyr Asn Ser Gly Asn Val Tyr Ile Met Ala Asp Lys GlnLys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn IleGlu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn ThrPro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr LeuSer Tyr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg AspHis Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr LeuGly Met Asp Glu Leu Tyr Lys 225 230 235 10 239 PRT Aequorea victoriaVARIANT (0)...(0) EYFP-H148Q 10 Met Val Ser Lys Gly Glu Glu Leu Phe ThrGly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn GlyHis Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr GlyLys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val ProTrp Pro Thr Leu Val Thr Thr 50 55 60 Phe Gly Tyr Gly Val Gln Cys Phe AlaArg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser AlaMet Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp AspGly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp ThrLeu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu AspGly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser GlnAsn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile LysVal Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val GlnLeu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 ProVal Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 195 200 205Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230235 11 720 DNA Aequorea victoria misc_feature (0)...(0) EGFP 11atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 12720 DNA Aequorea victoria misc_feature (0)...(0) EYFP 12 atggtgagcaagggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaaacggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctgaccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgaccaccttcggcta cggcgtgcag tgcttcgccc gctaccccga ccacatgaag 240 cagcacgacttcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacgacggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgcatcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagtacaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaaggtgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactaccagcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagctaccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagttcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 13 720 DNAAequorea victoria misc_feature (0)...(0) ECFP 13 atggtgagca agggcgaggagctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaggttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagttcatctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgacctggggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtccgccatgccc gaaggctacg tccaggagcg taccatcttc 300 ttcaaggacg acggcaactacaagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaagggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacatcagccacaac gtctatatca ccgccgacaa gcagaagaac 480 ggcatcaagg cccacttcaagatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacacccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgccctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgccgccgggatc actctcggca tggacgagct gtacaagtaa 720 14 720 DNA Aequoreavictoria misc_feature (0)...(0) EYFP-V68L/Q69K 14 atggtgagca agggcgaggagctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaagttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagttcatctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccttcggctacggcctgaag tgcttcgccc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtccgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaactacaagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaagggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaacagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaagatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacacccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagct accagtccgccctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgccgccgggatc actctcggca tggacgagct gtacaagtaa 720 15 714 DNA Aequoreavictoria misc_feature (0)...(0) YFP H148G 15 atgagtaaag gagaagaacttttcactgga gttgtcccaa ttcttgttga attagatggt 60 gatgttaatg ggcacaaattttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120 aaacttaccc ttaaatttatttgcactact ggaaaactac ctgttccatg gccaacactt 180 gtcactactt tcggttatggtcttcaatgc tttgcaagat acccagatca tatgaaacgg 240 catgactttt tcaagagtgccatgcccgaa ggttatgttc aggaaagaac tatatttttc 300 aaagatgacg ggaactacaagacacgtgct gaagtcaagt ttgaaggtga tacccttgtt 360 aatagaatcg agttaaaaggtattgatttt aaagaagatg gaaacattct tggacacaaa 420 ttggaataca actataactcaggcaatgta tacatcatgg cagacaaaca aaagaatgga 480 atcaaagtta acttcaaaattagacacaac attgaagatg gaagcgttca actagcagac 540 cattatcaac aaaatactccaattggcgat ggccctgtcc ttttaccaga caaccattac 600 ctgtcctatc aatctgccctttcgaaagat cccaacgaaa agagagacca catggtcctt 660 cttgagtttg taacagctgctgggattaca catggcatgg atgaactata caaa 714 16 714 DNA Aequorea victoriamisc_feature (0)...(0) YFP H148Q 16 atgagtaaag gagaagaact tttcactggagttgtcccaa ttcttgttga attagatggt 60 gatgttaatg ggcacaaatt ttctgtcagtggagagggtg aaggtgatgc aacatacgga 120 aaacttaccc ttaaatttat ttgcactactggaaaactac ctgttccatg gccaacactt 180 gtcactactt tcggttatgg tcttcaatgctttgcaagat acccagatca tatgaaacgg 240 catgactttt tcaagagtgc catgcccgaaggttatgttc aggaaagaac tatatttttc 300 aaagatgacg ggaactacaa gacacgtgctgaagtcaagt ttgaaggtga tacccttgtt 360 aatagaatcg agttaaaagg tattgattttaaagaagatg gaaacattct tggacacaaa 420 ttggaataca actataactc aggcaatgtatacatcatgg cagacaaaca aaagaatgga 480 atcaaagtta acttcaaaat tagacacaacattgaagatg gaagcgttca actagcagac 540 cattatcaac aaaatactcc aattggcgatggccctgtcc ttttaccaga caaccattac 600 ctgtcctatc aatctgccct ttcgaaagatcccaacgaaa agagagacca catggtcctt 660 cttgagtttg taacagctgc tgggattacacatggcatgg atgaactata caaa 714 17 720 DNA Aequorea victoria misc_feature(0)...(0) EYFP-H148G 17 atggtgagca agggcgagga gctgttcacc ggggtggtgcccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagggcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagctgcccgtgcc ctggcccacc 180 ctcgtgacca ccttcggcta cggcgtgcag tgcttcgcccgctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacgtccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtgaagttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggaggacggcaacat cctggggcac 420 aagctggagt acaactacaa cagcggcaac gtctatatcatggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgaggacggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccgtgctgctgcc cgacaaccac 600 tacctgagct accagtccgc cctgagcaaa gaccccaacgagaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggcatggacgagct gtacaagtaa 720 18 720 DNA Aequorea victoria misc_feature(0)...(0) EYFP-H148Q 18 atggtgagca agggcgagga gctgttcacc ggggtggtgcccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagggcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagctgcccgtgcc ctggcccacc 180 ctcgtgacca ccttcggcta cggcgtgcag tgcttcgcccgctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacgtccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtgaagttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggaggacggcaacat cctggggcac 420 aagctggagt acaactacaa cagccagaac gtctatatcatggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgaggacggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccgtgctgctgcc cgacaaccac 600 tacctgagct accagtccgc cctgagcaaa gaccccaacgagaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggcatggacgagct gtacaagtaa 720 19 255 PRT Aequorea victoria VARIANT (0)...(0)mito-ECFP 19 Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Phe Lys Arg Ser GlyIle 1 5 10 15 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val ProIle Leu 20 25 30 Val Glu Leu Asp Gly Asp Val Asn Gly His Arg Phe Ser ValSer Gly 35 40 45 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu LysPhe Ile 50 55 60 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu ValThr Thr 65 70 75 80 Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro AspHis Met Lys 85 90 95 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly TyrVal Gln Glu 100 105 110 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr LysThr Arg Ala Glu 115 120 125 Val Lys Phe Glu Gly Asp Thr Leu Val Asn ArgIle Glu Leu Lys Gly 130 135 140 Ile Asp Phe Lys Glu Asp Gly Asn Ile LeuGly His Lys Leu Glu Tyr 145 150 155 160 Asn Tyr Ile Ser His Asn Val TyrIle Thr Ala Asp Lys Gln Lys Asn 165 170 175 Gly Ile Lys Ala His Phe LysIle Arg His Asn Ile Glu Asp Gly Ser 180 185 190 Val Gln Leu Ala Asp HisTyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 195 200 205 Pro Val Leu Leu ProAsp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 210 215 220 Ser Lys Asp ProAsn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 225 230 235 240 Val ThrAla Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 245 250 255 20 255PRT Aequorea victoria VARIANT (0)...(0) mito-EYFP 20 Met Leu Ser Leu ArgGln Ser Ile Arg Phe Phe Lys Arg Ser Gly Ile 1 5 10 15 Met Val Ser LysGly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 20 25 30 Val Glu Leu AspGly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 35 40 45 Glu Gly Glu GlyAsp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 50 55 60 Cys Thr Thr GlyLys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 65 70 75 80 Phe Gly TyrGly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 85 90 95 Gln His AspPhe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 100 105 110 Arg ThrIle Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 115 120 125 ValLys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 130 135 140Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 145 150155 160 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn165 170 175 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp GlySer 180 185 190 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile GlyAsp Gly 195 200 205 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr GlnSer Ala Leu 210 215 220 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met ValLeu Leu Glu Phe 225 230 235 240 Val Thr Ala Ala Gly Ile Thr Leu Gly MetAsp Glu Leu Tyr Lys 245 250 255 21 323 PRT Aequorea victoria VARIANT(0)...(0) GT-EGFP 21 Met Arg Leu Arg Glu Pro Leu Leu Ser Gly Ala Ala MetPro Gly Ala 1 5 10 15 Ser Leu Gln Arg Ala Cys Arg Leu Leu Val Ala ValCys Ala Leu His 20 25 30 Leu Gly Val Thr Leu Val Tyr Tyr Leu Ala Gly ArgAsp Leu Ser Arg 35 40 45 Leu Pro Gln Leu Val Gly Val Ser Thr Pro Leu GlnGly Gly Ser Asn 50 55 60 Ser Ala Ala Ala Ile Gly Gln Ser Ser Gly Glu LeuArg Thr Gly Gly 65 70 75 80 Ala Met Asp Pro Met Val Ser Lys Gly Glu GluLeu Phe Thr Gly Val 85 90 95 Val Pro Ile Leu Val Glu Leu Asp Gly Asp ValAsn Gly His Lys Phe 100 105 110 Ser Val Ser Gly Glu Gly Glu Gly Asp AlaThr Tyr Gly Lys Leu Thr 115 120 125 Leu Lys Phe Ile Cys Thr Thr Gly LysLeu Pro Val Pro Trp Pro Thr 130 135 140 Leu Val Thr Thr Leu Thr Tyr GlyVal Gln Cys Phe Ser Arg Tyr Pro 145 150 155 160 Asp His Met Lys Gln HisAsp Phe Phe Lys Ser Ala Met Pro Glu Gly 165 170 175 Tyr Val Gln Glu ArgThr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 180 185 190 Thr Arg Ala GluVal Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 195 200 205 Glu Leu LysGly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 210 215 220 Lys LeuGlu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp 225 230 235 240Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile 245 250255 Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 260265 270 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr275 280 285 Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His MetVal 290 295 300 Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly MetAsp Glu 305 310 315 320 Leu Tyr Lys 22 323 PRT Aequorea victoria VARIANT(0)...(0) GT-EYFP 22 Met Arg Leu Arg Glu Pro Leu Leu Ser Gly Ala Ala MetPro Gly Ala 1 5 10 15 Ser Leu Gln Arg Ala Cys Arg Leu Leu Val Ala ValCys Ala Leu His 20 25 30 Leu Gly Val Thr Leu Val Tyr Tyr Leu Ala Gly ArgAsp Leu Ser Arg 35 40 45 Leu Pro Gln Leu Val Gly Val Ser Thr Pro Leu GlnGly Gly Ser Asn 50 55 60 Ser Ala Ala Ala Ile Gly Gln Ser Ser Gly Glu LeuArg Thr Gly Gly 65 70 75 80 Ala Met Asp Pro Met Val Ser Lys Gly Glu GluLeu Phe Thr Gly Val 85 90 95 Val Pro Ile Leu Val Glu Leu Asp Gly Asp ValAsn Gly His Lys Phe 100 105 110 Ser Val Ser Gly Glu Gly Glu Gly Asp AlaThr Tyr Gly Lys Leu Thr 115 120 125 Leu Lys Phe Ile Cys Thr Thr Gly LysLeu Pro Val Pro Trp Pro Thr 130 135 140 Leu Val Thr Thr Phe Gly Tyr GlyVal Gln Cys Phe Ala Arg Tyr Pro 145 150 155 160 Asp His Met Lys Gln HisAsp Phe Phe Lys Ser Ala Met Pro Glu Gly 165 170 175 Tyr Val Gln Glu ArgThr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 180 185 190 Thr Arg Ala GluVal Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 195 200 205 Glu Leu LysGly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 210 215 220 Lys LeuGlu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp 225 230 235 240Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile 245 250255 Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 260265 270 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr275 280 285 Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His MetVal 290 295 300 Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly MetAsp Glu 305 310 315 320 Leu Tyr Lys 23 263 PRT Aequorea victoria VARIANT(0)...(0) mito-YFP H148G 23 Met Leu Arg Thr Ser Ser Leu Phe Thr Arg ArgVal Gln Pro Ser Leu 1 5 10 15 Phe Arg Asn Ile Leu Arg Leu Gln Ser ThrSer Lys Gly Glu Glu Leu 20 25 30 Phe Thr Gly Val Val Pro Ile Leu Val GluLeu Asp Gly Asp Val Asn 35 40 45 Gly His Lys Phe Ser Val Ser Gly Glu GlyGlu Gly Asp Ala Thr Tyr 50 55 60 Gly Lys Leu Thr Leu Lys Phe Ile Cys ThrThr Gly Lys Leu Pro Val 65 70 75 80 Pro Trp Pro Thr Leu Val Thr Thr PheGly Tyr Gly Leu Gln Cys Phe 85 90 95 Ala Arg Tyr Pro Asp His Met Lys ArgHis Asp Phe Phe Lys Ser Ala 100 105 110 Met Pro Glu Gly Tyr Val Gln GluArg Thr Ile Phe Phe Lys Asp Asp 115 120 125 Gly Asn Tyr Lys Thr Arg AlaGlu Val Lys Phe Glu Gly Asp Thr Leu 130 135 140 Val Asn Arg Ile Glu LeuLys Gly Ile Asp Phe Lys Glu Asp Gly Asn 145 150 155 160 Ile Leu Gly HisLys Leu Glu Tyr Asn Tyr Asn Ser Gly Asn Val Tyr 165 170 175 Ile Met AlaAsp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile 180 185 190 Arg HisAsn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln 195 200 205 GlnAsn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 210 215 220Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 225 230235 240 Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr His245 250 255 Gly Met Asp Glu Leu Tyr Lys 260 24 263 PRT Aequorea victoriaVARIANT (0)...(0) mito-YFP H148Q 24 Met Leu Arg Thr Ser Ser Leu Phe ThrArg Arg Val Gln Pro Ser Leu 1 5 10 15 Phe Arg Asn Ile Leu Arg Leu GlnSer Thr Ser Lys Gly Glu Glu Leu 20 25 30 Phe Thr Gly Val Val Pro Ile LeuVal Glu Leu Asp Gly Asp Val Asn 35 40 45 Gly His Lys Phe Ser Val Ser GlyGlu Gly Glu Gly Asp Ala Thr Tyr 50 55 60 Gly Lys Leu Thr Leu Lys Phe IleCys Thr Thr Gly Lys Leu Pro Val 65 70 75 80 Pro Trp Pro Thr Leu Val ThrThr Phe Gly Tyr Gly Leu Gln Cys Phe 85 90 95 Ala Arg Tyr Pro Asp His MetLys Arg His Asp Phe Phe Lys Ser Ala 100 105 110 Met Pro Glu Gly Tyr ValGln Glu Arg Thr Ile Phe Phe Lys Asp Asp 115 120 125 Gly Asn Tyr Lys ThrArg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 130 135 140 Val Asn Arg IleGlu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 145 150 155 160 Ile LeuGly His Lys Leu Glu Tyr Asn Tyr Asn Ser Gln Asn Val Tyr 165 170 175 IleMet Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile 180 185 190Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln 195 200205 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 210215 220 Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg225 230 235 240 Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly IleThr His 245 250 255 Gly Met Asp Glu Leu Tyr Lys 260 25 265 PRT Aequoreavictoria VARIANT (0)...(0) mito-EYFP-H148G 25 Met Leu Arg Thr Ser SerLeu Phe Thr Arg Arg Val Gln Pro Ser Leu 1 5 10 15 Phe Arg Asn Ile LeuArg Leu Gln Ser Thr Met Val Ser Lys Gly Glu 20 25 30 Glu Leu Phe Thr GlyVal Val Pro Ile Leu Val Glu Leu Asp Gly Asp 35 40 45 Val Asn Gly His LysPhe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala 50 55 60 Thr Tyr Gly Lys LeuThr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu 65 70 75 80 Pro Val Pro TrpPro Thr Leu Val Thr Thr Phe Gly Tyr Gly Val Gln 85 90 95 Cys Phe Ala ArgTyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys 100 105 110 Ser Ala MetPro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys 115 120 125 Asp AspGly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp 130 135 140 ThrLeu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp 145 150 155160 Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser Gly Asn 165170 175 Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe180 185 190 Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala AspHis 195 200 205 Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu LeuPro Asp 210 215 220 Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys AspPro Asn Glu 225 230 235 240 Lys Arg Asp His Met Val Leu Leu Glu Phe ValThr Ala Ala Gly Ile 245 250 255 Thr Leu Gly Met Asp Glu Leu Tyr Lys 260265 26 265 PRT Aequorea victoria VARIANT (0)...(0) mito-EYFP-H148Q 26Met Leu Arg Thr Ser Ser Leu Phe Thr Arg Arg Val Gln Pro Ser Leu 1 5 1015 Phe Arg Asn Ile Leu Arg Leu Gln Ser Thr Met Val Ser Lys Gly Glu 20 2530 Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp 35 4045 Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala 50 5560 Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu 65 7075 80 Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Gly Tyr Gly Val Gln 8590 95 Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys100 105 110 Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe PheLys 115 120 125 Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe GluGly Asp 130 135 140 Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp PheLys Glu Asp 145 150 155 160 Gly Asn Ile Leu Gly His Lys Leu Glu Tyr AsnTyr Asn Ser Gln Asn 165 170 175 Val Tyr Ile Met Ala Asp Lys Gln Lys AsnGly Ile Lys Val Asn Phe 180 185 190 Lys Ile Arg His Asn Ile Glu Asp GlySer Val Gln Leu Ala Asp His 195 200 205 Tyr Gln Gln Asn Thr Pro Ile GlyAsp Gly Pro Val Leu Leu Pro Asp 210 215 220 Asn His Tyr Leu Ser Tyr GlnSer Ala Leu Ser Lys Asp Pro Asn Glu 225 230 235 240 Lys Arg Asp His MetVal Leu Leu Glu Phe Val Thr Ala Ala Gly Ile 245 250 255 Thr Leu Gly MetAsp Glu Leu Tyr Lys 260 265 27 972 DNA Aequorea victoria misc_feature(0)...(0) GT-ECFP 27 atgaggcttc gggagccgct cctgagcggc gccgcgatgccaggcgcgtc cctacagcgg 60 gcctgccgcc tgctcgtggc cgtctgcgct ctgcaccttggcgtcaccct cgtttactac 120 ctggctggcc gcgacctgag ccgcctgccc caactggtcggagtctccac accgctgcag 180 ggcggctcga acagtgccgc cgccatcggg cagtcctccggggagctccg gaccggaggg 240 gccatggatc ccatggtgag caagggcgag gagctgttcaccggggtggt gcccatcctg 300 gtcgagctgg acggcgacgt aaacggccac aggttcagcgtgtccggcga gggcgagggc 360 gatgccacct acggcaagct gaccctgaag ttcatctgcaccaccggcaa gctgcccgtg 420 ccctggccca ccctcgtgac caccctgacc tggggcgtgcagtgcttcag ccgctacccc 480 gaccacatga agcagcacga cttcttcaag tccgccatgcccgaaggcta cgtccaggag 540 cgtaccatct tcttcaagga cgacggcaac tacaagacccgcgccgaggt gaagttcgag 600 ggcgacaccc tggtgaaccg catcgagctg aagggcatcgacttcaagga ggacggcaac 660 atcctggggc acaagctgga gtacaactac atcagccacaacgtctatat caccgccgac 720 aagcagaaga acggcatcaa ggcccacttc aagatccgccacaacatcga ggacggcagc 780 gtgcagctcg ccgaccacta ccagcagaac acccccatcggcgacggccc cgtgctgctg 840 cccgacaacc actacctgag cacccagtcc gccctgagcaaagaccccaa cgagaagcgc 900 gatcacatgg tcctgctgga gttcgtgacc gccgccgggatcactctcgg catggacgag 960 ctgtacaagt aa 972 28 768 DNA Aequorea victoriamisc_feature (0)...(0) mito-EYFP 28 atgctgagcc tgcgccagag catccgcttcttcaagcgca gcggcatcat ggtgagcaag 60 ggcgaggagc tgttcaccgg ggtggtgcccatcctggtcg agctggacgg cgacgtaaac 120 ggccacaagt tcagcgtgtc cggcgagggcgagggcgatg ccacctacgg caagctgacc 180 ctgaagttca tctgcaccac cggcaagctgcccgtgccct ggcccaccct cgtgaccacc 240 ttcggctacg gcgtgcagtg cttcgcccgctaccccgacc acatgaagca gcacgacttc 300 ttcaagtccg ccatgcccga aggctacgtccaggagcgca ccatcttctt caaggacgac 360 ggcaactaca agacccgcgc cgaggtgaagttcgagggcg acaccctggt gaaccgcatc 420 gagctgaagg gcatcgactt caaggaggacggcaacatcc tggggcacaa gctggagtac 480 aactacaaca gccacaacgt ctatatcatggccgacaagc agaagaacgg catcaaggtg 540 aacttcaaga tccgccacaa catcgaggacggcagcgtgc agctcgccga ccactaccag 600 cagaacaccc ccatcggcga cggccccgtgctgctgcccg acaaccacta cctgagctac 660 cagtccgccc tgagcaaaga ccccaacgagaagcgcgatc acatggtcct gctggagttc 720 gtgaccgccg ccgggatcac tctcggcatggacgagctgt acaagtaa 768 29 972 DNA Aequorea victoria misc_feature(0)...(0) GT-EGFP 29 atgaggcttc gggagccgct cctgagcggc gccgcgatgccaggcgcgtc cctacagcgg 60 gcctgccgcc tgctcgtggc cgtctgcgct ctgcaccttggcgtcaccct cgtttactac 120 ctggctggcc gcgacctgag ccgcctgccc caactggtcggagtctccac accgctgcag 180 ggcggctcga acagtgccgc cgccatcggg cagtcctccggggagctccg gaccggaggg 240 gccatggatc ccatggtgag caagggcgag gagctgttcaccggggtggt gcccatcctg 300 gtcgagctgg acggcgacgt aaacggccac aagttcagcgtgtccggcga gggcgagggc 360 gatgccacct acggcaagct gaccctgaag ttcatctgcaccaccggcaa gctgcccgtg 420 ccctggccca ccctcgtgac caccctgacc tacggcgtgcagtgcttcag ccgctacccc 480 gaccacatga agcagcacga cttcttcaag tccgccatgcccgaaggcta cgtccaggag 540 cgcaccatct tcttcaagga cgacggcaac tacaagacccgcgccgaggt gaagttcgag 600 ggcgacaccc tggtgaaccg catcgagctg aagggcatcgacttcaagga ggacggcaac 660 atcctggggc acaagctgga gtacaactac aacagccacaacgtctatat catggccgac 720 aagcagaaga acggcatcaa ggtgaacttc aagatccgccacaacatcga ggacggcagc 780 gtgcagctcg ccgaccacta ccagcagaac acccccatcggcgacggccc cgtgctgctg 840 cccgacaacc actacctgag cacccagtcc gccctgagcaaagaccccaa cgagaagcgc 900 gatcacatgg tcctgctgga gttcgtgacc gccgccgggatcactctcgg catggacgag 960 ctgtacaagt aa 972 30 972 DNA Aequorea victoriamisc_feature (0)...(0) GT-EYFP 30 atgaggcttc gggagccgct cctgagcggcgccgcgatgc caggcgcgtc cctacagcgg 60 gcctgccgcc tgctcgtggc cgtctgcgctctgcaccttg gcgtcaccct cgtttactac 120 ctggctggcc gcgacctgag ccgcctgccccaactggtcg gagtctccac accgctgcag 180 ggcggctcga acagtgccgc cgccatcgggcagtcctccg gggagctccg gaccggaggg 240 gccatggatc ccatggtgag caagggcgaggagctgttca ccggggtggt gcccatcctg 300 gtcgagctgg acggcgacgt aaacggccacaagttcagcg tgtccggcga gggcgagggc 360 gatgccacct acggcaagct gaccctgaagttcatctgca ccaccggcaa gctgcccgtg 420 ccctggccca ccctcgtgac caccttcggctacggcgtgc agtgcttcgc ccgctacccc 480 gaccacatga agcagcacga cttcttcaagtccgccatgc ccgaaggcta cgtccaggag 540 cgcaccatct tcttcaagga cgacggcaactacaagaccc gcgccgaggt gaagttcgag 600 ggcgacaccc tggtgaaccg catcgagctgaagggcatcg acttcaagga ggacggcaac 660 atcctggggc acaagctgga gtacaactacaacagccaca acgtctatat catggccgac 720 aagcagaaga acggcatcaa ggtgaacttcaagatccgcc acaacatcga ggacggcagc 780 gtgcagctcg ccgaccacta ccagcagaacacccccatcg gcgacggccc cgtgctgctg 840 cccgacaacc actacctgag ctaccagtccgccctgagca aagaccccaa cgagaagcgc 900 gatcacatgg tcctgctgga gttcgtgaccgccgccggga tcactctcgg catggacgag 960 ctgtacaagt aa 972 31 762 DNAAequorea victoria misc_feature (0)...(0) mito-YFP H148G 31 atgctgagcctgcgccagag catccgcttc ttcaagcgca gcggcatcat gagtaaagga 60 gaagaacttttcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg 120 cacaaattttctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt 180 aaatttatttgcactactgg aaaactacct gttccatggc caacacttgt cactactttc 240 ggttatggtcttcaatgctt tgcaagatac ccagatcata tgaaacggca tgactttttc 300 aagagtgccatgcccgaagg ttatgttcag gaaagaacta tatttttcaa agatgacggg 360 aactacaagacacgtgctga agtcaagttt gaaggtgata cccttgttaa tagaatcgag 420 ttaaaaggtattgattttaa agaagatgga aacattcttg gacacaaatt ggaatacaac 480 tataactcaggcaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac 540 ttcaaaattagacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa 600 aatactccaattggcgatgg ccctgtcctt ttaccagaca accattacct gtcctatcaa 660 tctgccctttcgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta 720 acagctgctgggattacaca tggcatggat gaactataca aa 762 32 762 DNA Aequorea victoriamisc_feature (0)...(0) mito-YFP H148Q 32 atgctgagcc tgcgccagagcatccgcttc ttcaagcgca gcggcatcat gagtaaagga 60 gaagaacttt tcactggagttgtcccaatt cttgttgaat tagatggtga tgttaatggg 120 cacaaatttt ctgtcagtggagagggtgaa ggtgatgcaa catacggaaa acttaccctt 180 aaatttattt gcactactggaaaactacct gttccatggc caacacttgt cactactttc 240 ggttatggtc ttcaatgctttgcaagatac ccagatcata tgaaacggca tgactttttc 300 aagagtgcca tgcccgaaggttatgttcag gaaagaacta tatttttcaa agatgacggg 360 aactacaaga cacgtgctgaagtcaagttt gaaggtgata cccttgttaa tagaatcgag 420 ttaaaaggta ttgattttaaagaagatgga aacattcttg gacacaaatt ggaatacaac 480 tataactcag gcaatgtatacatcatggca gacaaacaaa agaatggaat caaagttaac 540 ttcaaaatta gacacaacattgaagatgga agcgttcaac tagcagacca ttatcaacaa 600 aatactccaa ttggcgatggccctgtcctt ttaccagaca accattacct gtcctatcaa 660 tctgcccttt cgaaagatcccaacgaaaag agagaccaca tggtccttct tgagtttgta 720 acagctgctg ggattacacatggcatggat gaactataca aa 762 33 768 DNA Aequorea victoria misc_feature(0)...(0) mito-EYFP-H148G 33 atgctgagcc tgcgccagag catccgcttc ttcaagcgcagcggcatcat ggtgagcaag 60 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcgagctggacgg cgacgtaaac 120 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatgccacctacgg caagctgacc 180 ctgaagttca tctgcaccac cggcaagctg cccgtgccctggcccaccct cgtgaccacc 240 ttcggctacg gcgtgcagtg cttcgcccgc taccccgaccacatgaagca gcacgacttc 300 ttcaagtccg ccatgcccga aggctacgtc caggagcgcaccatcttctt caaggacgac 360 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcgacaccctggt gaaccgcatc 420 gagctgaagg gcatcgactt caaggaggac ggcaacatcctggggcacaa gctggagtac 480 aactacaaca gcggcaacgt ctatatcatg gccgacaagcagaagaacgg catcaaggtg 540 aacttcaaga tccgccacaa catcgaggac ggcagcgtgcagctcgccga ccactaccag 600 cagaacaccc ccatcggcga cggccccgtg ctgctgcccgacaaccacta cctgagctac 660 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatcacatggtcct gctggagttc 720 gtgaccgccg ccgggatcac tctcggcatg gacgagctgtacaagtaa 768 34 768 DNA Aequorea victoria misc_feature (0)...(0)mito-EYFP-H148Q 34 atgctgagcc tgcgccagag catccgcttc ttcaagcgcagcggcatcat ggtgagcaag 60 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcgagctggacgg cgacgtaaac 120 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatgccacctacgg caagctgacc 180 ctgaagttca tctgcaccac cggcaagctg cccgtgccctggcccaccct cgtgaccacc 240 ttcggctacg gcgtgcagtg cttcgcccgc taccccgaccacatgaagca gcacgacttc 300 ttcaagtccg ccatgcccga aggctacgtc caggagcgcaccatcttctt caaggacgac 360 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcgacaccctggt gaaccgcatc 420 gagctgaagg gcatcgactt caaggaggac ggcaacatcctggggcacaa gctggagtac 480 aactacaaca gccagaacgt ctatatcatg gccgacaagcagaagaacgg catcaaggtg 540 aacttcaaga tccgccacaa catcgaggac ggcagcgtgcagctcgccga ccactaccag 600 cagaacaccc ccatcggcga cggccccgtg ctgctgcccgacaaccacta cctgagctac 660 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatcacatggtcct gctggagttc 720 gtgaccgccg ccgggatcac tctcggcatg gacgagctgtacaagtaa 768 35 5 PRT Homo sapiens 35 Lys Lys Lys Arg Lys 1 5 36 26 PRTHomo sapiens 36 Met Leu Arg Thr Ser Ser Leu Phe Thr Arg Arg Val Gln ProSer Leu 1 5 10 15 Phe Arg Asn Ile Leu Arg Leu Gln Ser Thr 20 25 37 4 PRTHomo sapiens 37 Lys Asp Glu Leu 1 38 4 PRT Aequorea victoria VARIANT(0)...(0) Linker sequence 38 Arg Ser Gly Ile 1

What is claimed is:
 1. A polynucleotide comprising a nucleotide sequenceencoding a functional engineered fluorescent protein whose emissionintensity changes as pH varies between 5 and 10, wherein said functionalengineered fluorescent protein is selected from the group consisting ofa) a protein comprising the substitutions S65G/S72A/T203Y/H231L withinthe amino acid sequence of Aequorea green fluorescent protein (SEQ IDNO:2), b) a protein comprising the substitutionsS65G/V68L/Q69K/S72A/T203Y/H231L within the amino acid sequence ofAequorea green fluorescent protein (SEQ ID NO:2), c) a proteincomprising the substitutionsK26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L within the amino acidsequence of Aequorea green fluorescent protein (SEQ ID NO:2), d) aprotein comprising the substitution H148G within the amino acid sequenceof Aequorea green fluorescent protein (SEQ ID NO: 2), and e) a proteincomprising the substitutions S65G/V68L/S72A/Q80R/H148Q/T203Y orS65G/S72A/H148Q/T203Y/H231L within the amino acid sequence of Aequoreagreen fluorescent protein (SEQ ID NO:2), where the amino acid sequenceof said functional engineered fluorescent protein is at least 95%homologous to the amino acid sequence of SEQ ID NO:2.
 2. Thepolynucleotide of claim 1, where the protein comprising the substitutionH148G within the amino acid sequence of Aequorea green fluorescentprotein (SEQ ID NO:2) is selected from the group of proteins consistingof the protein comprising the substitutionsS65G/V68L/S72A/Q80R/H148G/T203Y within the amino acid sequence ofAequorea green fluorescent protein (SEQ ID NO:2) and the proteincomprising the substitutions S65G/S72A/H148G/T203Y/H231L within theamino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2).3. An expression vector comprising at least one expression controlsequence operatively linked to a polynucleotide of claim
 1. 4. Arecombinant host cell comprising the expression vector of claim
 3. 5.The recombinant host cell of claim 4, wherein the recombinant host cellis a prokaryotic cell.
 6. The recombinant host cell of claim 4, whereinthe recombinant host cell is a eukaryotic cell.
 7. A kit useful for thedetection of the pH in a sample, the kit comprising carrier meanscontaining one or more containers comprising a first containercontaining a polynucleotide comprising a nucleotide sequence encoding afunctional engineered fluorescent protein whose emission intensitychanges as pH varies between 5 and 10, wherein said functionalengineered fluorescent protein is selected from the group consisting ofa) a protein comprising the substitutions S65G/S72A/T203Y/H231L withinthe amino acid sequence of Aequorea green fluorescent protein (SEQ IDNO:2), b) a protein comprising the substitutionsS65G/V68L/Q69K/S72A/T203Y/H231L within the amino acid sequence ofAequorea green fluorescent protein (SEQ ID NO:2), c) a proteincomprising the substitutionsK26R/F64L/S65T/Y66W/N146I/M153T/V163A/N164H/H231L within the amino acidsequence of Aequorea green fluorescent protein (SEQ ID NO:2), d) aprotein comprising the substitution H148G within the amino acid sequenceof Aequorea green fluorescent protein (SEQ ID NO:2), and e) a proteincomprising the substitutions S65G/V68L/S72A/Q80R/H148Q/T203Y orS65G/S72A/H148Q/T203Y/H231L within the amino acid sequence of Aequoreagreen fluorescent protein (SEQ ID NO:2), where the amino acid sequenceof said functional engineered fluorescent protein is at least 95%homologous to the amino acid sequence of SEQ ID NO:2.
 8. The kit ofclaim 7, where the protein comprising the substitution H148G within theamino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2)is selected from the group of proteins consisting of the proteincomprising the substitutions S65G/V68L/S72A/Q80R/H148G/T203Y within theamino acid sequence of Aequorea green fluorescent protein (SEQ ID NO:2)and the protein comprising the substitutions S65G/S72A/H148G/T203Y/H231Lwithin the amino acid sequence of Aequorea green fluorescent protein(SEQ ID NO:2).